Stabilized bcl9 peptides for treatment of aberrant wnt signaling

ABSTRACT

Methods for treating cancer with a stabilized BCL9 peptide are encompassed, wherein the stabilized peptide comprises a portion of the HD2 domain of the BCL9 protein containing a hydrocarbon crosslinker generated using α,α-disubstituted amino acids.

This application is a continuation of and claims priority to U.S.application Ser. No. 17/215,698, filed Mar. 29, 2021, which is adivisional of and claims priority to U.S. application Ser. No.15/766,268, filed Apr. 5, 2018, now U.S. Pat. No. 10,961,290, which is aNational Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2016/055589, filed Oct. 5, 2016, which claimspriority to U.S. Provisional Application No. 62/237,489, filed Oct. 5,2015. The disclosures of the prior applications are incorporated byreference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named 44574-0004003_SL_ST26.xml. The XMLfile, created on Sep. 30, 2022, is 334,549 bytes in size. The materialin the XML file is hereby incorporated by reference in its entirety.

FIELD

Disclosed here are polypeptides derived from the HD2 domain of humanB-cell CLL/lymphoma 9 (BCL9) protein and variants thereof, as well astheir use in the diagnosis, prevention, and/or treatment of a disease ordisorder. Also disclosed are methods of generating such polypeptides andvariants thereof.

BRIEF DESCRIPTION

β-catenin is a multifunctional protein of critical importance tocellular homeostasis and processes such as embryogenesis, epithelialcell growth, and organ regeneration. However, aberrant β-cateninsignaling can lead to changes in transcriptional activation that canallow tumor growth and development. β-catenin is normally phosphorylatedand targeted for degradation by the axin complex, but unphosphorylatedβ-catenin can accumulate if there is stimulation of the Wnt signalingpathway. Under conditions when the Wnt signaling pathway is activated,β-catenin binds to lymphoid enhancer factor/T cell factor (LEF/TCF) andis translocated into the nucleus to stimulate transcription of Wnttarget genes (see Clevers and Nusse, Cell 149:1192-1205 (2012)), such asc-myc and CD44, that play roles in tumorigenesis.

Aberrant activation of the Wnt/β-catenin pathway has been shown in avariety of human cancers (see Thakur and Mishra, J Cell Mol Med17(4):449-456 (2013)). Overactive β-catenin signaling can result inuncontrolled cellular proliferation within tumors, as well as affectingsurvival of cancer cells. In addition, β-catenin can support tumormetastasis by increasing the migratory and invasive capabilities ofcancers cells. Up to 90% of all cases of sporadic colorectal cancers areassociated with constitutive activation of Wnt signaling.

BCL9 is a protein known to be required for efficient β-catenin-mediatedtranscription in mammalian cells (see de la Roche et al., BMC Cancer8:199 (2008)). BCL9 binds via its HD2 domain to β-catenin, and mutationsin the HD2 domain disrupt the association of BCL9 with β-catenin.

Agents that selectively target β-catenin without impacting other Wntsignalling pathways are attractive targets for treatment of patientswith cancer. Numerous approaches to develop agents that inhibitβ-catenin activity have been employed (see Thakur and Mishra 2013). Manysmall molecule compounds targeted to inhibit β-catenin have shownpromising efficacy in animal models, but more tailored and selectiveapproaches to inhibiting β-catenin activation are needed.

One approach to selectively modulate β-catenin activity is the use ofstabilized peptides comprising portions of proteins known to interactwith and regulate the function of β-catenin. Stabilized peptides havenumerous advantages over wild-type peptides as potential therapeutics,including increased helical content, proteolytic stability, andincreased binding affinity for a target receptor (see Kim et al., NatProtocols 6(6):761-771 (2011)). One β-catenin interactor for whichstabilized peptides have been investigated is BCL9, which binds toβ-catenin through its HD2 domain. However, attempts to use hydrocarbonlinkers (i.e. “hydrocarbon stapling”) to generate stabilized peptidescomprising portions of the HD2 domain of BCL9 have been hampered by theexpense of synthesis and low yield (see Kawamoto, PhD Dissertation inMedicinal Chemistry, Univ of Michigan (2010)). Other investigators havedeveloped stabilized peptides of BCL9 wherein the α-helix of the HD2domain of the BCL9 protein is stabilized via a hydrocarboncrosslinker(s) generated via ring-closing metathesis (RCM) (seeUS20140113857).

While these stabilized peptides have had some success in the art, thereremains a need for stabilized peptides that are more capable ofmodulating immune response and therefore have improved in vivo efficacyin treating a tumor.

Disclosed herein are polypeptides derived from the HD2 domain of humanB-cell CLL/lymphoma 9 (BCL9) protein. In some embodiments, thepolypeptide has a length of 7-14 amino acids. In various embodiments,the polypeptide is stabilized via one or more hydrocarbon crosslinkers,resulting in a construct interchangeably referred as a “stabilizedpolypeptide” or “stapled polypeptide.” In various embodiments, thepolypeptide derived from the HD2 domain is capable of undergoing areaction to form one or more hydrocarbon crosslinkers, and is referredas an “unstapled polypeptide.”

In some embodiments, the polypeptide capable of undergoing a reaction toform one or more hydrocarbonds comprises any sequence selected fromTable 1 or a variant thereof. In some embodiments, the Xaa₁, Xaa₂, Xaa₃and/or Xaa₄ listed in Table 1 are each an α,α-disubstituted amino acid.In some embodiments, a hydrocarbon crosslinker is present between Xaa₃and Xaa₄. In some embodiments, a hydrocarbon crosslinker is presentbetween Xaa₁ and Xaa₂. In some embodiments, a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂ and a hydrocarbon crosslinker is presentbetween Xaa₃ and Xaa₄.

In some embodiments, the polypeptide comprises one or morehydrocarbonds, comprising any sequence selected from Table 1 or avariant thereof. In some embodiments, Xaa₁, Xaa₂, Xaa₃, and/or Xaa₄ areeach alanine or an an α,α-disubstituted alanine, a first hydrocarboncrosslinker is present between Xaa₁ and Xaa₂, and/or a secondhydrocarbon crosslinker is present between Xaa₃ and Xaa₄.

In some embodiments, the polypeptide is capable of undergoing a reactionto form one or more hydrocarbon crosslinkers and comprises or consistsof LQTLRXaa₁IQRXaa₂L (SEQ ID NO: 1) or a variant thereof. In someembodiments, Xaa₁ and Xaa₂ are each α,α-disubstituted amino acids.

In some embodiments, the polypeptide is capable of undergoing a reactionto form one or more hydrocarbon crosslinkers and comprises or consistsof Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ ID NO: 2) or a variant thereof. In someembodiments, Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are each α,α-disubstituted aminoacids. In some embodiments, Xaa₁ and Xaa₂ are each an α,α-disubstitutedamino acid and a hydrocarbon crosslinker is present between Xaa₃ andXaa₄. In some embodiments, Xaa₃ and Xaa₄ are each an α,α-disubstitutedamino acid and a hydrocarbon crosslinker is present between Xaa₁ andXaa₂.

In some embodiments, the polypeptide comprises a hydrocarbon crosslinkerand comprises or consists of LQTLRXaa₁IQRXaa₂L (SEQ ID NO: 1) or avariant thereof. In some embodiments, Xaa₁ and Xaa₂ are each alanine anda hydrocarbon crosslinker is present between Xaa₁ and Xaa₂.

In some other embodiments, the polypeptide comprises a hydrocarboncrosslinker and comprises or consists of Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQID NO: 2) or a variant thereof. In certain embodiments, Xaa₁, Xaa₂, Xaa₃and Xaa₄ are each alanine and a first hydrocarbon crosslinker is presentbetween Xaa₁ and Xaa₂ and a second hydrocarbon crosslinker is presentbetween Xaa₃ and Xaa₄.

In some embodiments, the α,α-disubstituted amino acid is an α-methyl,α-alkenyl amino acid. In some embodiments, the α-methyl, α-alkenyl aminoacid is selected from (S)-2-(4′-pentenyl)alanine,(R)-2-(4′-pentenyl)alanine, (S)-2-(7′-octenyl)alanine, and(R)-2-(7′-octenyl)alanine.

In some embodiments, the hydrocarbon linker is selected from—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2- and—CH2-CH2-CH2-CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-. In some embodiments, thehydrocarbon crosslinker has an S-configuration on at least one end, oron both ends. In some embodiments, the hydrocarbon crosslinker has anR-configuration on at least one end, or on both ends. In someembodiments, the hydrocarbon crosslinker has an S-configuration on oneend and an R-configuration on the other end.

In further embodiments, the N-terminus and/or C-terminus of thepolypeptide or variant are further modified. In some embodiments, theN-terminus is modified with an acetyl group. In some embodiments, theC-terminus is modified with one, two, or more units of β-alanine,2-Naphthylalanine, and/or 2-Naphthylalanine, and optionally linked toone, two, or more units of β-alanine, and wherein the carboxyl group ofthe C-terminus modification is optionally further modified with an NH₂group. In some embodiments, the N-terminus and/or C-terminusmodification further comprise a fluorenylmethyloxycarbonyl (Fmoc) group.

In some embodiments, the stapled polypeptide or variant described hereinhas one or more improved biological functions as compared to anunstapled wild-type human BLC9 HD2 domain or a fragment of a wild-typehuman BLC9 HD2 domain. When the stapled polypeptide or variant isadministered to a subject and/or contacted with a target cell, thepolypeptide or variant may be capable of exhibiting one or more improvedbiological functions selected from: inhibiting binding of BCL9 toβ-catenin, inhibiting canonical Wnt/β-catenin signaling, decreasingregulatory T cell survival, decreasing expression of VEGF in a tumor,increasing CD4⁺ T cell and/or CD8⁺ T cell infiltration into a tumor,increasing T helper 17 (Th17) cell infiltration into a tumor, decreasingdendritic cells in a tumor, having a half-file (T_(1/2)) greater than atleast 2 hours, inducing a tumor microenvironment favoring an immunereaction, and inhibiting tumor growth, cancer stem cell proliferation,and/or tumor metastasis.

Also disclosed herein are pharmaceutical compositions, comprising thestapled polypeptide or variant derived from the HD2 domain of human BLC9protein and a pharmaceutically acceptable carrier. In some embodiments,the pharmaceutical composition comprises at least one additional agent,such as a checkpoint inhibitor, an EGFR inhibitor, a VEGF inhibitor, aVEGFR inhibitor, or an anti-cancer drug.

Also disclosed herein are methods of making and using the claimedstapled polypeptide or variant, e.g., in inhibiting, reducing,preventing, and/or treating a cancer in a subject. In some embodiments,the methods of using the claimed polypeptide or variant encompassinhibiting binding of BCL9 to β-catenin in a subject, as well asinhibiting canonical Wnt/β-catenin signaling in a subject.

Also disclosed herein are biomarkers used in monitoring treatmentefficacy and/or selecting a subject to be treated with the claimedstapled polypeptide or variant. In some embodiments, the subjectadministered with the claimed polypeptide or variant is also treatedwith an additional therapeutic agent, radiation, and/or chemotherapy. Akit for making and using the claimed stapled polypeptide or variant isalso disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show the structures of the stabilized peptidederived from the HD2 domain of BCL9 protein, corresponding to SEQ ID NO:103 and SEQ ID NO: 104, respectively. The stabilized polypeptide of SEQID NO: 103, referred as “WX-024” in this application. The stabilizedpolypeptide of SEQ ID NO: 104 is referred as “WX-035” in thisapplication. These peptides have an alkenyl linker(s) generated betweenα-methyl, α-alkenyl amino acids to stabilize the α-helix of the HD2domain of the BCL9 protein.

FIG. 2A depicts a domain mapping strategy that systematically walksthrough the HD2 domain of human BCL9 protein (SEQ ID NOS 114-130, 107,131 and 106, respectively, in order of appearance). FIG. 2B showsresults of a cell viability assay using increasing amounts of eachdomain fragment generated based on the domain mapping strategy depictedin FIG. 2A. ICG001, a known small molecule inhibitor of canonicalWnt/β-catenin signaling, was used as a positive control. The cellviability assay was performed using CellTiterGlo assay (Promega) withColo320DM cells. FIG. 2C shows results of a Wnt transcription assayusing increasing amounts of each domain fragment generated in FIG. 2A.ICG001 was used as a positive control. The Wnt transcription assay wasperformed using GeneBlazer Wnt Reporter Assay (Invitrogen) with HCT116cells.

FIG. 3 depicts results of a cell viability assay comparing WX-020 andSAH-BCL9 #1. The cell viability assay was performed using CellTiterGloassay (Promega) with Colo320DM cells.

FIG. 4A depicts results of a cell viability assay comparing WX-020 andfour different stabilized polypeptides derived from WX-020, includingWX-024. The cell viability assay was performed using a CellTiterGloassay (Promega) with HCT116 cells. ICG001 was used as a positivecontrol. FIG. 4B depicts results of the acetate salt form of WX-024tested in a cell-based Wnt transcription inhibition assay (IC₅₀=292 nM).FIG. 4C shows the effect of the hydrochloric salt also tested in thesame Wnt transcription inhibition assay (IC₅₀=313 nM).

FIGS. 5A, 5B, 5C, and 5D show results of a GeneBlazer Wnt Reporter Assay(Invitrogen) testing WX-021, WX-024, and ICG001. FIG. 5A provides aschematic of the reporter assay where binding of BCL9 to β-catenindrives expression of a beta-lactamase (BLA) reporter gene in CellSensor™LEF/TCF-bla HCT-116 cells (Invitrogen) (Abbreviations: β-cat, β-catenin;TF, transcription factor). A peptide corresponding to amino acids in theHD2 domain, such as WX-024, can inhibit binding of BCL9 to β-catenin.FIGS. 5B, 5C, and 5D depict results of the reporter assay using WX-021,WX-024, and ICG001, respectively. The IC₅₀ values of WX-021, WX-024, andICG001 calculated from the results of this assay were 764 nM, 191 nM,and 1060 nM, respectively. FIG. 5E shows that WX-024 also showed betterin vitro potency targeting Wnt/β-catenin transcription than LGK-974(IC₅₀>10 μM), which was expected, since LGK-974 targets extracellularWnt signaling and does not directly inhibit β-cat transcription.

FIG. 6A and FIG. 6B show results of a homogenous time resolvedfluorescence (HTRF) assay (Cisbio) measuring WX-024 (conjugated withbiotin) binding to β-catenin. FIG. 6A provides a schematic of the HTRFassay, showing that the assay measures binding of a biotinylated BCL9peptide (such as biotinylated WX-024) to β-catenin by assessingfluorescence resonance energy transfer (FRET) (Abbreviations: ab,antibody; SA, streptavidin). FIG. 6B shows the K_(D) determination forbinding of WX-024 to β-catenin based on the HTRF assay (K_(D)=4.21 nM).

FIG. 7 shows cell viability data comparing WX-024 with ICG001 (aWnt/β-catenin pathway inhibitor) using a CellTiterGlo assay (Promega) inHCT116 cells. The IC₅₀ values of WX-024 and ICG001 calculated based onthis assay were 1.92 μM and 2.17 μM, respectively.

FIG. 8A shows results of a cell viability assay testing WX-024, ICG001,and LGK-974 (*P<0.05, WX-024 vs ICG001, LGK-974). Colo320DM cells wereused in this cell viability assay. FIG. 8B shows results of the samecell viability assay testing WX-024 and Erlotinib. FIG. 8C shows resultsof the same cell viability assay comparing 5-FU treatment alone and acombination of 5-FU and WX-024 (*P<0.05).

FIG. 9A and FIG. 9B show pharmacokinetic (PK) data for WX-024 withintravenous (i.v.) administration at 1 mg/kg or 5 mg/kg andintraperitoneal (i.p.) administration at 5 mg/kg to male ICR mice (anoutbred strain). Blood samples were collected at 15 min, 1, 2, 4, 6, 8,12, and 24 hours post-dosing and analyzed via liquid chromatography-massspectrometry (LC-MS), as shown in FIG. 9A. From the plasma concentrationdata shown in FIG. 9A, the maximum observed concentration (C_(max)),terminal half-life (T_(1/2)), total body clearance (CL), volume ofdistribution (V_(z)), area under the curve from the time of dosing tothe last measurable concentration (AUC_(0-t)), area under the curve fromthe time of dosing extrapolated to infinity (AUC_(0-inf)), andbioavailability were calculated. These pharmacokinetic parameters aresummarized in FIG. 9B.

FIG. 10A shows PK data for WX-024 measured in female Balb/c mice. Eachdata point represents N=2. Mice were administered 5 mg/kg, 10 mg/kg, or15 mg/kg intravenously, 5 mg/kg intramuscularly, 10 mg/kgintraperitoneally, or 10 mg/kg subcutaneously. Blood samples werecollected at 15 min, 1, 2, 4, 6, 8, 12, and 24 hours (and 36 and 48hours post-dose for intramuscular, subcutaneous, and intraperitonealroutes) post-dosing and analyzed via liquid chromatography-massspectrometry (LC-MS). FIG. 10B and FIG. 10C summarize the meanpharmacokinetic parameters calculated from this experiment.

FIG. 11 shows body weight data of female Balb/c nude mice at 6-8 weeksof age inoculated with Colo320DM tumor cells and treated with a vehicleor WX-024. Mice were inoculated with Colo320DM tumor cells (5×10⁶), andtumors were allowed to develop for 14 days, after which treatments wereapplied. The vehicle and 5 mg/kg treatment groups were dosed throughoutthe study by i.v. injection with no apparent change in body weight overthe study. After 3 days of i.v. dosing of 15 mg/kg WX-024, mice in thisgroup exhibited body weight loss. Therefore, on Day 7, the 10 and 15mg/kg groups were switched to i.p. dosing for the rest of the treatmentperiod. Following this switch to i.p. dosing, body weight in the 15mg/kg group stabilized for the remainder of the study.

FIG. 12A and FIG. 12B depict tumor growth changes caused by vehicle orWX-024 during the experiment described for FIG. 11 . As shown in FIG.12A, tumor volumes (expressed in mm³) were measured using calipers atdays 0, 3, 5, 7, 9, 11, and 14 of the treatment period (*P<0.05,compared to vehicle group by Kruskal-Wallis analysis). FIG. 12B showsthe average tumor mass of each treatment group at the conclusion of thestudy (day 22; *P<0.05, compared to vehicle group).

FIG. 13A shows intestinal histology of BALB/c nude mice at theconclusion of the Colo320DM xenograft model after 22 days of treatment,as described for FIG. 11 . Samples from vehicle, 10 mg/kg, and 15 mg/kgWX-024 treatment groups are depicted. FIG. 13B shows Axin2 staining ofthe intestine samples collected in FIG. 13A.

FIG. 14A shows 40×-magnification of CD44 staining of tumor samplescollected from the experiment described for FIG. 11 . Theimmunochemistry score of each sample is shown at the upper right cornerof each image. FIG. 14B summarizes the average immunochemistry score ofeach treatment group.

FIG. 15A shows VEGFA staining of tumor samples collected from theexperiment described for FIG. 11 . The immunochemistry score of eachsample is shown at the upper right corner of each image. FIG. 15Bsummarizes the average immunochemistry score of each treatment group.

FIGS. 16A, 16B, 16C, and 16D depict results from a CellTiter Glo assay(Promega) measuring CT26 cell (ATCC) growth in vitro when treated withWX-024 or doxorubicin. FIG. 16A shows the growth rate of untreated cellsfrom day 1 (D1) to day 5 (D5). FIG. 16B and FIG. 16C show the growthinhibition rate in WX-024 or doxorubicin-treated cells as measured inthis assay, respectively. FIG. 16D summarizes the concentration ofagents producing 50% inhibition of growth (IC₅₀), the minimuminhibition, the maximum inhibition, and the relative IC₅₀ that is themedian value calculated based on each curve depicted in FIG. 16B andFIG. 16C.

FIG. 17A shows the average tumor growth of two groups of mice treatedwith either vehicle or 20 mg/kg WX-024. Each mouse received anintraperitoneal injection of its assigned treatment daily for 14 days. ABalb/c syngeneic mouse model bearing CT26 colon cancer cells was used inthis experiment. Each data point represents the average of eight miceper group. FIG. 17B shows the average tumor growth inhibition (TGI) rateof WX-024 treatment. The TGI at day 3 was 89.2% while the TGI at day 12was 70.0%.

FIG. 18A and FIG. 18B show the tumor growth of individual animalsmonitored in the experiment described for FIG. 17 . FIG. 18A representsthe tumor growth of each mouse treated with a vehicle while FIG. 18Brepresents the tumor growth of each mouse treated with WX-024.

FIGS. 19A and 19B show CD4⁺ T cell counts in blood samples collectedfrom the mice depicted in FIG. 17 . The blood samples were collected atthe conclusion of the experiment (day 14). FIG. 19A shows CD4⁺ T cellcounts presented as the percentage of total cells in each blood sample.FIG. 19B shows relative T cell count per total cells in each bloodsample.

FIG. 20A and FIG. 20B show CD8⁺ T cell counts in blood samples collectedfrom the mice depicted in FIG. 17 . The blood samples were collected atthe conclusion of the experiment (day 14). FIG. 20A shows CD8⁺ T cellcounts presented as the percentage of total cells in each blood sample.FIG. 20B shows relative T cell count per total cells in each bloodsample.

FIG. 21A depicts the percentage of regulatory T cells in CD4⁺ T cellpopulations in tumor samples collected from the experiment described inFIG. 17 . FIG. 21B depicts the ratio between CD8⁺ T cells and regulatoryT cells in the same tumor samples. FIG. 21C and FIG. 21D showrepresentative FACS analysis of samples obtained from blood and tumor,respectively, tested in this experiment.

FIG. 22A shows active β-catenin staining of tumor samples collected fromthe experiment described in FIG. 17 . The top four panels show thestaining images of four samples collected from a vehicle treated groupwhile the bottom four panels show the staining images of four samplescollected from a WX-024 treated group. FIG. 22B represents the averageimmunohistochemical score of each treatment group. FIG. 22C showsrepresentative staining of CD44 in tumor samples collected from the sameexperiment.

FIG. 23A shows PD-L1 staining of tumor samples collected from theexperiment described in FIG. 17 . The top four panels show the stainingimages of four samples collected from a vehicle treated group while thebottom four panels show the staining images of four samples collectedfrom a WX-024 treated group. FIG. 23B represents the averageimmunohistochemical score of each treatment group.

FIG. 24 shows tumor growth changes caused by vehicle or WX-024 inC57BL/6 mice inoculated with B16 cells (1×10³). The mice wereintraperitoneally administered with either a vehicle or 25 mg/kg WX-024daily for 14 consecutive days (n=6 per group).

FIGS. 25A, 25B, and 25C depict T cell infiltration into tumor caused byWX-024 treatment. Female 5 weeks of age C57BL/6 mice were inoculatedwith B16 cells (1×10³). When the average tumor volume of the inoculatedmice reached 100 mm³, the mice were divided into two groups andintraperitoneally administered with either a vehicle or 25 mg/kg WX-024for 12 consecutive days. At the conclusion of the experiment, tumorsamples were collected and stained with either CD4 or CD8. FIG. 25Ashows CD4⁺ T cell counts presented as a percentage of total tumor cells.FIG. 25B shows CD8⁺ T cell counts presented as a percentage of totaltumor cells. FIG. 25C represents CD4⁺ or CD8⁺ T cell counts presented asa percentage of total tumor cells.

FIG. 26A and FIG. 26B depict the effects of WX-024 and WX-039 ondendritic cells in present in tumor. C57BL/6 mice were inoculated withB16 cells. When the average tumor volume of the inoculated mice reached100 mm³, the mice were divided into two groups and intraperitoneallyadministered with a vehicle, 20 mg/kg WX-024, or 60 mg/kg WX-039 for 12consecutive days. At the conclusion of the experiment, tumor sampleswere collected and stained for dendritic cell markers and T helper 17cell markers. FIG. 26A shows myeloid dendritic cells (mDC) count whileFIG. 26B shows plastic dendritic cells (pDC) count. (* p<0.05; ***p<0.0001, compared to vehicle control group; One-Way ANOVA). FIG. 26Cshows CD194+ or CD196+ T cells presented as a percentage of total tumorcells. FIG. 26D shows a representative FACS analysis of a tumor sampletested in this experiment.

FIG. 27A depicts the average total flux of orthotopic mice treated witheither a vehicle or 15 mg/kg WX-024. Balb/c female nude mice wereinjected with NCI-H1299-Luc cells (5×10⁶ in 50 μL in PBS) and treatedintravenously with a vehicle or WX-024 for 10 consecutive days. 3 miceper group were tested. The total flux indicates the luminescence photonflux signal generated by tumor cells in the mice. FIG. 27B depicts thetotal flux of each mouse monitored in the same experiment. FIG. 27Cshows the bioluminescence image of each mouse monitored in thisexperiment.

FIG. 28A shows immunohistochemical staining of selective biomarkers infour samples derived from colorectal cancer patients (scale bar: 100μm). Each sample was stained with H&E (Haemotoxylin and Eosin), IgG,β-catenin, BCL9, c-Myc, and CD44. Colorectal cancer sample #8 wasselected for further examination. FIG. 28B shows the effect of WX-024 onpatient-derived xenograft NOD/SCID mice inoculated with cells derivedfrom patient #8. After two weeks of inoculation, the mice were dividedinto two groups (N=8 per group) and treated with a vehicle or 15 mg/kgWX-024 intravenously for the following four weeks. The average tumorgrowth inhibition at day 31 by WX-024 was 75.6%.

FIG. 29A depict β-catenin and BCL9 staining of tumor samples collectedfrom the experiment described in FIG. 28 . The immunohistochemical scoreof each image is shown at the top right corner of the image. FIG. 29Bdepict CD44 staining of tumor samples collected from the experimentdescribed in FIG. 28 .

FIGS. 30A, 30B, and 30C depict the safety profile of WX-024 assessed infemale balb/c mice. The mice were treated intravenously with vehicle, 10mg/kg, 15 mg/kg, or 20 mg/kg WX-024 for 14 consecutive days (n=6). FIG.30A shows H&E staining of major organs harvested from the mice treatedwith either vehicle or 10 mg/kg WX-024 at the conclusion of theexperiment (day 14). FIG. 30B shows the average body weight of eachtreatment group throughout the experiment. FIG. 30C shows the completeblood cell count profiles of a vehicle treated group and a 20 mg/kgWX-024 treated group.

FIG. 31A and FIG. 31B show the toxicokinetic analysis of female balb/cmice intraperitoneally treated with vehicle, 10 mg/kg, 15 mg/kg, or 20mg/kg WX-024 for 14 consecutive days. FIG. 31A shows the average WX-024concentration in whole blood after the dosing on day 1. FIG. 31B showsthe average WX-024 concentration in whole blood after the dosing on day14.

FIG. 32 shows results of a cell viability assay testing WX-024 andWX-035. Colo320DM cells were used in this experiment.

FIG. 33A and FIG. 33B show PK data for WX-035 administered to male ICRmice (an outbred strain). The mice were treated with either 1 mg/kg, or5 mg/kg WX-035 intravenously or 5 mg/kg WX-035 intraperitoneally. Bloodsamples from each mouse were collected at 15 min, 1, 2, 4, 6, 8, 12, and24 hours post administration. FIG. 33A shows the concentration of WX-035in the samples analyzed via liquid chromatography-mass spectrometry(LC-MS). From the concentration data, the maximum observed concentration(C_(max)), terminal half-life (T_(1/2)), total body clearance (CL),volume of distribution (V_(z)), area under the curve from the time ofdosing to the last measurable concentration (AUC_(0-t)), area under thecurve from the time of dosing extrapolated to infinity (AUC_(0-inf)),and bioavailability were calculated, as shown in FIG. 33B.

FIG. 34A and FIG. 34B show PK data for WX-035 administered to femalebalb/c mice (n=3). The mice were either intravenously orintraperitoneally administered with 30 mg/kg WX-035. Blood samples fromeach mouse were collected at 5 min, 15 min, 30 min, 1, 2, 4, 6, 8, 12,24, 36, 48, and 72 hours post administration. FIG. 34A shows theconcentration of WX-035 in the samples analyzed via liquidchromatography-mass spectrometry (LC-MS). From the concentration data,the time to reach the maximum concentration (T_(max)), the maximumobserved concentration (C_(max)), terminal half-life (T_(1/2)), areaunder the curve from the time of dosing to the last measurableconcentration (AUC_(last)), area under the curve from the time of dosingextrapolated to infinity (AUC_(INF)), and bioavailability (F) werecalculated, as shown in FIG. 34B.

FIG. 35A shows the effect of WX-035 on tumor growth inhibition tested inmale balb/c syngeneic animal models. The mice were inoculated with CT26cells (1×10³). When the average tumor volume reached 100 mm³, the micewere divided into three groups and treated intraperitoneally with avehicle, 20 mg/kg WX-035, or 1 mg/kg trametinib for 7 consecutive days(n=7). FIG. 35B compares the average tumor growth inhibition by WX-035and by trametinib in this experiment. At day 7, the average tumor growthinhibition by WX-035 was 88.44%.

FIGS. 36A, 36B, 36C, and 36D show the in vivo effect of WX-035 on T regcells and other types of T cell populations. At the conclusion of theexperiment depicted in FIG. 35 , tumor samples were collected from avehicle treated group and a WX-035 treated group. FIG. 36A shows totalCD45⁺ T cells as a percentage of total tumor cells. FIG. 36B shows totalCD4⁺ or CD8⁺ T cells as a percentage of total tumor cells. FIG. 36Cshows total CD25⁺/Foxp3⁺ T cells. FIG. 36D depicts the ratio betweenCD8⁺ T cells and CD25⁺/Foxp3⁺ T cells in both treatment groups. FIG. 36Eshows LGR5 staining of intestine samples collected in this experiment.

FIG. 37 depicts H&E staining of major organs harvested from female 6weeks of age balb/c nude mice intravenously treated with vehicle, 30mg/kg, or 40 mg/kg WX-035 for 7 consecutive days (scale bar: 100 μm).

FIG. 38A and FIG. 38B show the effects of WX-035 and WX-037 in a cellviability assay using CT26.WT cells. FIG. 38C summarizes the in vitroprofiles of each polypeptide. Ab IC₅₀ indicates the absoluteconcentration of an inhibitor where the cell viability is reduced byhalf.

FIG. 39A and FIG. 39B show PK data for WX-029 administered to femalebalb/c nude mice (N=2). 1 mg/kg of WX-029 was administered to each mouseintravenously. FIG. 39A shows the plasma concentration measured after 15min, 1, 2, or 4 hours of administration. FIG. 39B summarizes thepharmacokinetic profiles calculated using the plasma concentrationresults shown in FIG. 39A.

FIG. 40A and FIG. 40B show PK data for WX-036 administered to femalebalb/c nude mice (N=2). 1 mg/kg WX-036 was administered to each mouseintravenously. FIG. 40A shows the plasma concentration measured after 15min, 1, 2, or 4 hours of administration. FIG. 40B summarizes thepharmacokinetic profiles calculated using the plasma concentrationresults shown in FIG. 40A.

FIG. 41A and FIG. 41B show PK data for WX-039 administered to femalebalb/c nude mice (N=2). WX-039 was administered to each mouse at 5 mg/kgintravenously, 5 mg/kg intraperitoneally, or 10 mg/kg subcutaneously.FIG. 41A shows the plasma concentration measured after 15 min, 1, 2, 4,8, 24, 36, 48, and 72 hours of administration. FIG. 41B summarizes thepharmacokinetic profiles calculated using the plasma concentrationresults shown in FIG. 41A.

FIG. 42A and FIG. 42B show PK data for WX-036 administered to femalebalb/c nude mice (N=2). WX-040 was administered to each mouse at 30mg/kg intravenously or 40 mg/kg intraperitoneally. FIG. 42A shows theplasma concentration measured after 15 min, 1, 2, 4, 8, 24, 36, 48, and72 hours of administration. FIG. 42B summarizes the pharmacokineticprofiles calculated using the plasma concentration results shown in FIG.42A.

FIG. 43A shows the effect of WX-039 on tumor growth inhibition tested in5 weeks old female C57BL/6 mice inoculated with B16F10 cells (N=3). Thetumor growth inhibition rate assessed during the experiment is shown inFIG. 43B. FIG. 43C shows the body weight change during the experimentdepicted in FIG. 43A.

DETAILED DESCRIPTION

A. BCL-9, β-Catenin, and Wnt Signaling

Aberrant activation of Wnt signaling is implicated in a variety ofcancers, as tumors can become dependent on Wnt signaling for growth andsurvival (see Grossmann et al. PNAS. 109(44):17942-17947 (2012)). Up to90% of all cases of sporadic colorectal cancers are associated withconstitutive activation of Wnt signaling.

β-catenin is a protein that can engage in protein-protein interactionsthat stimulate Wnt signaling leading to changes in transcriptionalactivation that can allow tumor growth and development. β-catenin isnormally phosphorylated and targeted for degradation by the Axincomplex. If there is stimulation of the Wnt signaling pathway,unphosphorylated β-catenin accumulates and binds to lymphoid enhancerfactor/T cell factor (LEF/TCF) and is translocated into the nucleus tostimulate transcription of Wnt target genes (see Thakur 2013). Wnttarget genes include c-myc and CD44, which are upregulated genes intumor models. BCL9 is a protein required for efficientβ-catenin-mediated transcription in mammalian cells (see de la Roche etal., BMC Cancer 8:199 (2008)).

“Canonical” Wnt/β-catenin signaling is a pathway activated by Wntligands binding to the Frizzled family of cell-surface receptors, whichthen regulate expression and intracellular localization of β-catenin. Inthe absence of Wnt ligands, β-catenin is phosphorylated andubiquitinated within a destruction complex composed of adenomatouspolyposis coli (APC), glycogen synthase kinase-3 (GSK-3), caseinkinase-1 (CK1) and Axin, and targeted for degradation in aproteasome-dependent manner. In the presence of Wnt ligands,ubiquitination of β-catenin within the complex is suppressed, leading tosaturation of phosphorylated β-catenin, which is then stabilized andtranslocated to the nucleus. There, phosphorylated β-catenin engagesnuclear T-cell factor (TCF) transcription factors, such as LymphoidEnhancer Factor/3 (LEF/TCF), to induce expression of genes that promotecell proliferation, migration, and survival, including c-Myc28 andCyclin D.

Several molecules, including BCL9 and its homologue B-cell lymphoma9-like (B9L), have been shown to be co-activators for Wnt/β-catenintranscription. The formation of a complex consisting of TCF, β-catenin,and BCL9 (or B9L) enhances β-catenin-dependent Wnt transcriptionalactivity. In normal cells, this transcriptional pathway is turned offwhen Wnt ligands uncouple from their receptors. However, a variety ofloss-of-function mutations in APC and Axin, as well as activatingmutations in β-catenin itself, enable β-catenin to escape thedestruction complex and accumulate in the nucleus. Such inappropriatepersistence of β-catenin promotes oncogenesis in a wide range of commonhuman epithelial cancers, including hepatocellular, breast, colorectal,and hematological malignancies such as multiple myeloma. In addition,active β-catenin signaling results in T-cell exclusion, specificallyCD8⁺ T-cells, which leads to therapy resistance and shorter patientsurvival times. Thus, blocking Wnt signaling by targeting β-cat mayoffer a powerful way to treat CRC, potentially preventing both tumorinitiation and metastasis. See Spranger et al., Nature 523: 231-235(2015).

Similar to other transcription factors, the development of selective,non-toxic β-catenin inhibitors and their translation to the clinic haveproven to be a considerable challenge, as β-catenin interacts with themajority of its protein partners through the same binding surface. Thus,Wnt pathway inhibitors targeting this common binding surface haveexhibited significant adverse effects in animal and clinical trials.There are only a few drugs targeting β-catenin in clinical trials,including PRI-724 (Eisai Pharmaceuticals; Phase II), LGK974 (Novartis;Phase I), and OMP-54F28 and OMP-18R5 (OncoMed/Bayer; Phase I). Inaddition, disruption of LEF/TCF interaction through small molecule andpeptide inhibitors of β-cat can have serious side effects, includingsevere bone marrow hypoplasia, anemia, and generalized wasting oftreated mice—likely a result of disrupting homeostatic Wnt signaling innormal hematopoietic and intestinal stem cells. Such therapeuticlimitations may derive from disruption of β-catenin-TCF andβ-catenin-E-cadherin interactions, which can affect epithelial tissueintegrity. Furthermore, biological agents targeting the Frizzledreceptor (OMP-54F28 and OMP-18R5) have shown significant bone marrowtoxicity during clinical trials. The Wnt ligand is essential forWnt/β-cat activation, but APC and β-catenin mutations in cancer cellscould induce downstream transcription without Wnt ligand activation, soblocking Wnt secretion cannot inhibit endogenous oncogenic Wnt activitydue to APC and β-catenin mutations induced downstream genetranscription. LGK974 only targets a small patient population, asidentified by certain biomarkers. PRI-724, a small molecule inhibitor,is under phase II trials with daily infusion, but more than once-weeklyintravenous (IV) dosing exhibits characteristics undesirable anduntenable for clinical development.

Traditionally, Wnt signaling pathways include three different types ofsignaling: a canonical Wnt signaling pathway where Wnt regulates varioustranscriptional target genes through a β-catenin dependent manner; anoncanonical Wnt signaling pathway mainly involved in planer cellpolarity, where Wnt may function independently of β-catenin; and anoncanonical Wnt/calcium pathway regulating an intracellular calciumlevel. In the present application, “canonical Wnt signaling” isinterchangeably referred as “canonical Wnt/β-catenin signaling” or “Wntsignaling.” As described herein, canonical Wnt/β-catenin signaling mayrefer to pathway components that control the amount of β-catenin in apatient or sample, e.g., by modulating the stability of β-catenin. Insome embodiments, canonical Wnt/β-catenin signaling comprises pathwaycomponents that transcriptionally modulate one or more genes such asc-myc, ccnd1, cd44, LGR5, VEGFA, AXIN2, and LEF1. In some embodiments,canonical Wnt/β-catenin signaling comprises pathway components that aremodulated by the interaction between β-catenin and BCL9. In someembodiments, canonical Wnt/β-catenin signaling comprises one or moregenes that are transcriptionally controlled by the interaction betweenβ-catenin and BCL9. The one or more genes controlled by the interactionbetween β-catenin and BCL9 may include c-myc, ccnd1, cd44, LGR5, VEGFA,AXIN2, and LEF1. In some embodiments, canonical Wnt/β-catenin signalingcomprises one or more proteins, the transcriptional expressions of whichare modulated by the interaction between β-catenin and BCL9. Thosecomponents may include, for example, c-Myc, Cyclin D1, CD44, LGR5,VEGFA, AXIN2, and LEF1.

B. Stabilized BCL-9 Peptides

Stabilized peptides have been shown to confer advantages such asincreased helical content, proteolytic stability, and increased bindingaffinity for a target receptor (see Kim 2011). In particular, α-helixdomains are known to be amenable to stabilization.

1. Polypeptide Derived from BCL9 HD2 Domain

The HD2 domain of BCL9 mediates the binding of BCL9 to β-catenin, and sofar, the HD2 domain is the only domain of BLC9 shown to bind toβ-catenin in cells (see de la Roche 2008). The HD2 domain forms anα-helix, and thus polypeptides derived from the HD2 domain of BCL9 inwhich the α-helix has been stabilized may be appropriate for inhibitingthe interaction of BCL9 with β-catenin (Sampietro et al., MolecularCell, 24, 293-300 (2006)).

In one embodiment, mapping the activity of portions of the HD2 domain ofthe BCL9 protein is done to determine active regions, which areinterchangeably referred as core functional domains. In one embodiment,a peptide containing a portion of the HD2 domain of the BCL9 isstructurally constrained. In certain embodiments, this structuralconstraint stabilizes an α-helix of the BCL9 peptide.

In some embodiments, a polypeptide described herein is derived from theHD2 domain of human BCL9 protein. As used herein, the terms“polypeptide,” “peptide,” and “protein” are used interchangeably andrefer to a polymer comprising two or more amino acids bonded together toform a chain. The term “polypeptide derived from the HD2 domain of humanBCL9 protein” encompasses the full-length HD2 domain of human BCL9protein and fragments of such HD2 domain. Also encompassed here arevariants of the full-length HD2 domain or the fragments. The sequence ofthe full-length HD2 domain of human BCL9 protein (SEQ ID NO: 3) is shownin Table 1. A polypeptide derived from the HD2 domain of human BCL9protein, or a variant thereof, can be stapled or stabilized as disclosedherein.

In some embodiments, the polypeptide described herein comprises thefull-length HD2 domain of human BCL9 protein. In some embodiments, thepolypeptide described herein comprises a fragment and/or variant of theHD2 domain of human BCL9 protein. In a certain embodiment, thepolypeptide derived from the HD2 domain of human BCL9 protein comprisesa fragment of the HD2 domain of human BCL9 protein, which is furthermodified by substituting one or more amino acids with other naturallyoccurring amino acids or non-naturally occurring amino acids. In someembodiment, the polypeptide derived from the HD2 domain of human BCL9protein is capable of undergoing a reaction to form one or morehydrocarbon crosslinkers. As used herein, the polypeptide capable ofundergoing a reaction to form one or more hydrocarbon crosslinkers maybe referred as an “unstapled polypeptide.” In some embodiments, apolypeptide described herein comprises one or more non-naturallyoccurring amino acids. In some embodiments, the non-naturally occurringamino acid is norleucine. In some embodiments, the non-naturallyoccurring amino acid is an α,α-disubstituted amino acid. In someembodiments, the non-naturally occurring amino acid is an α-methyl,α-alkenyl amino acid. In some embodiment, the non-naturally occurringamino acid is a chiral molecule, comprising a chiral center with eitherS- or R-configuration. In some embodiment, the non-naturally occurringamino acid is selected from (S)-2-(4′-pentenyl)alanine,(R)-2-(4′-pentenyl)alanine, (S)-2-(7′-octenyl)alanine, and(R)-2-(7′-octenyl)alanine.

As used herein, a polypeptide derived from the HD2 domain of human BCL9protein also encompasses a polypeptide that has undergone a reaction toform one or more hydrocarbon crosslinkers and thus comprises one or morehydrocarbon crosslinkers. As used herein, the polypeptide comprising oneor more hydrocarbon crosslinkers may be referred as a “stabilizedpolypeptide” or a “stapled polypeptide.” In some embodiments, thehydrocarbon crosslinker has a length of 2-15 carbons. In someembodiments, the hydrocarbon crosslinker has a length of 5-11 carbons.In some embodiments, the hydrocarbon crosslinker has a length of 7-11carbons. In some embodiments, the hydrocarbon crosslinker has a lengthof 7-15 carbons. In some embodiments, the hydrocarbon crosslinker has alength of 8-11 carbons. In some embodiments, the hydrocarbon crosslinkerhas a length of 7 or 8 or 9 or 10 or 11 carbons, or more. In someembodiments, the hydrocarbon crosslinker is selected from—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂— and—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—.

In some embodiments, the stapled polypeptides described herein arecapable of inhibiting the binding of BCL9 to β-catenin in vitro and/orin vivo. In some embodiments, a polypeptide derived from the HD2 domainof human BCL9 protein has one or more improved biological functions ascompared to an unstapled wild-type HD2 domain of human BCL9 protein oras compared to a fragment of an unstapled wild-type HD2 domain. The oneor more biological functions may be selected from one or more of: (1)inhibiting binding of BCL9 to β-catenin; (2) inhibiting canonicalWnt/β-catenin signaling; (3) decreasing regulatory T cell survival; (4)decreasing expression of VEGF in a tumor; (5) increasing CD4⁺ T cell andCD8⁺ T cell infiltration into a tumor; (6) increasing T helper 17 (Th17)cells in a tumor; (7) decreasing dendritic cells in a tumor; (8) havinga half-life (T½) greater than at least 2 hours when administrated to asubject; (9) inducing a tumor microenvironment favoring an immunereaction; and (10) inhibiting tumor growth, cancer stem cellproliferation, and/or tumor metastasis.

The present disclosures encompass stapled peptides comprising a variantof a wild-type HD2 domain of human BCL9 protein, or a variant of afragment of an unstapled wild-type HD2 domain, that retains one or morebiological functions of the wild-type polypeptide. A “variant” as usedherein in connection with the polypeptide described herein refers to apolypeptide that differs from a given polypeptide in amino acid sequenceand/or chemical structure, but retains one or more biological functionsof the given polypeptide (i.e., the polypeptide described herein). Forinstance, the variant may retain one or more biological functions of apolypeptide derived from the HD2 domain of human BCL9 protein such ase.g., the ability to bind I3-catenin, inhibiting canonical Wnt/β-cateninsignaling, and/or inhibit binding of BCL9 to β-catenin.

The variant polypeptide described herein may have one or more amino acidadditions (e.g., insertion), deletions, and/or substitutions from thegiven polypeptide, as long as it retains the functional propertiesmentioned above. In some embodiments, the variant polypeptide describedherein may have 1-30, 1-20, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2, or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid additions (e.g.,insertion), deletions, and/or substitutions from the wild-typepolypeptide, including all integers in between these ranges.

In some embodiments, the variant comprises conservative substitution ofone or more amino acids of a given polypeptide. A conservativesubstitution of an amino acid, i.e., replacing an amino acid with adifferent amino acid of similar properties (e.g., hydrophilicity, anddegree and distribution of charged regions), typically involves a minorchange and therefore does not significantly alter the biologicalactivity of the polypeptide. These minor changes may be identified byconsidering the hydropathic index of amino acids based on aconsideration of the hydrophobicity and charge of the amino acid. Aminoacids of similar hydropathic indexes and hydrophilicity values can besubstituted and still retain protein function. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function depend on the relative similarity of the aminoacids, and particularly the side chains of those amino acids, asrevealed by the hydrophobicity, hydrophilicity, charge, size, and otherproperties.

In some embodiments, the variant comprises substitution of one or moreamino acids of a wild-type polypeptide or fragment by a non-naturallyoccurring amino acid. In some embodiments, the non-naturally occurringamino acid is norleucine. In some embodiments, the non-naturallyoccurring amino acid is an α,α-disubstituted amino acid. In someembodiments, the non-naturally occurring amino acid is an α-methyl,α-alkenyl amino acid. In some embodiment, the non-naturally occurringamino acid is a chiral molecule, comprising a chiral center with eitheran S- or R-configuration. In some embodiment, the non-naturallyoccurring amino acid is selected from (S)-2-(4′-pentenyl)alanine,(R)-2-(4′-pentenyl)alanine, (S)-2-(7′-octenyl)alanine, and(R)-2-(7′-octenyl)alanine.

The term “variant” also includes a polypeptide that has a certainpercent homology, such as, e.g., at least about 50%, 60%, 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) to awild-type polypeptide or fragment. As used herein, the term percent (%)homology defines the percentage of residues in the amino acid sequencesof the variant and the given polypeptide that are identical afteraligning the sequences and other spacing, e.g., using the BLASTalignment software.

In some embodiments, the variant comprises a polypeptide that ischemically and/or post-translationally modified in a manner differentfrom the wild-type polypeptide or fragment, but retains one or morebiological functions as described above. For instance, the variant maycomprise one or more amino acids that are post-transitionally modifiedby e.g., phosphorylation, acetylation, methylation, ubiquitination,SUMOylation, or other post-translational modifications known in the art.The variant may also comprise one or more chemical modifications, e.g.,one or more amino acid side chains that are modified or substituted witha different chemical moiety. As used herein, the term “variant” alsoencompasses a polypeptide that is identical to a given polypeptide inamino acid sequence, but having a different hydrocarbon crosslinker. Theterm “variant” may also refer to a polypeptide that is identical to agiven polypeptide in amino acid sequence and chemical structure, buthaving a different chirality.

2. Structure of Polypeptide Derived from BCL9 HD2 Domain

In some embodiments, a polypeptide derived from the HD2 domain of humanBCL9 protein has a length of 7-22 amino acids. In some embodiments, thepolypeptide has a length of 7-14 amino acids. In some embodiments, thepolypeptide has a length of 7 or 8 amino acids. In some embodiments, thepolypeptide has a length of 10-14 amino acids. In some embodiments, thepolypeptide has a length of 11 or 12 amino acids.

In some embodiments, a polypeptide derived from the HD2 domain of humanBCL9 protein has a length of 7-14 amino acids and comprises any sequenceselected from Table 1. In some embodiments, the polypeptide derived fromthe HD2 domain of human BCL9 protein has a length of 7 or 8 amino acidsand comprises any sequence selected from Table 1. In some embodiments, apolypeptide derived from the HD2 domain of human BCL9 protein has alength of 10-14 amino acids and comprises any sequence selected fromTable 1. In some embodiments, a polypeptide derived from the HD2 domainof human BCL9 protein has a length of 10 or 11 amino acids and comprisesany sequence selected from Table 1. The polypeptides described hereinalso encompass variants of a polypeptide selected from Table 1 thatretains one or more biological functions of the polypeptide. In variousembodiments, the selected polypeptide is stabilized, e.g., by comprisingone or more hydrocarbon cross-link. In Table 1, B indicates norleucine.

TABLE 1 Polypeptides derived from the HD2 domain of human BCL9 proteinSEQ Generic Corresponding ID Polypep position N-terminus C-terminus NO:ID Amino Acid Sequence within BCL9 Modification modificiation  3 HD2PDGLSQEQLEHRERSLQT 348-377 N/A N/A LRDIQRMLFPDE  4 WX-LSQEQLEHRERSLXaa₁T 351-374 Ac NH₂ 001 LRXaa₂IQRBLF  5 WX- LXaa₁QEQXaa₂E351-357 Ac NH₂ 002  6 WX- SXaa₁EQLXaa₂H 352-358 Ac NH₂ 003  7 WX-QXaa₁QLEXaa₂R 353-359 Ac NH₂ 004  8 WX- EXaa₁LEHXaa₂E 354-360 Ac NH₂ 005 9 WX- QXaa₁EHRXaa₂R 355-361 Ac NH₂ 006 10 WX- LXaa₁HREXaa₂S 356-362 AcNH₂ 007 11 WX- EXaa₁RERXaa₂L 357-363 Ac NH₂ 008 12 WX- HXaa₁ERSXaa₂Q358-364 Ac NH₂ 009 13 WX- RXaa₁RSLXaa₂T 359-365 Ac NH₂ 010 14 WX-EXaa₁SLQXaa₂L 360-366 Ac NH₂ 011 15 WX- RXaa₁LQTXaa₂R 361-367 Ac NH₂ 01216 WX- SXaa₁QTLXaa₂D 362-368 Ac NH₂ 013 17 WX- LXaa₁TLRXaa₂I 363-369 AcNH₂ 014 18 WX- QXaa₁LRDXaa₂Q 364-370 Ac NH₂ 015 19 WX- TXaa₁RDIXaa₂R365-371 Ac NH₂ 016 20 WX- LXaa₁DIQXaa₂B 366-372 Ac NH₂ 017 21 WX-RXaa₁IQRXaa₂L 367-373 Ac NH₂ 018 22 WX- DXaa₁QRBXaa₂F 368-374 Ac NH₂ 01923 WX- LRXaa₁IQRXaa₂L 366-373 Ac NH₂ 020 24 WX- LRXaa₁IQRXaa₂L 366-373Ac 2-Nal-ß- 021 Ala-ß- Ala-NH₂ 25 WX- LRXaa₁IQRXaa₂L 366-373 Ac ß-Ala-ß-022 Ala-NH₂ 26 WX- LRXaa₁IQRXaa₂L 366-373 Ac 2-Nal-NH₂ 023 27 WX-LQTLRXaa₁IQRXaa₂L 363-373 Ac 2-Nal-NH₂ 024 28 WX- LQTLRXaa₁IQRXaa₂L363-373 Ac 2-Nal-B- 029 Ala-ß- Ala- GRKKRRQRR RPQ 29 WX-Xaa₁LQXaa₂LRXaa₃IQR 362-373 Ac 2-Nal-ß- 035 Xaa₄L Ala-ß- Ala-NH₂ 30 WX-Xaa₁LQXaa₂LRXaa₃IQR 362-373 Ac 2-Nal-ß- 036 Xaa₄L Ala-ß- Ala- GRKKRRQRRRPQ 31 WX- Xaa₁DQXaa₂DRXaa₃DQR 362-374 Ac ß-Ala-ß- 037 Xaa₄DH Ala-NH₂ 32WX- Xaa₁LEXaa₂LRXaa₃IER 362-373 Ac 2-Nal-ß- 038 Xaa₄L Ala-ß- Ala-NH₂ 33WX- RXaa₁LQXaa₂LRXaa₃IQ 361-373 Ac 2-Nal-ß- 039 RXaa₄L Ala-ß- Ala-NH₂ 34WX- LQXaa₁LRDIQRXaa₂L 363-373 Ac 2-Nal-ß- 040 Ala-ß- Ala-NH₂

In a certain embodiment, a polypeptide described herein is capable ofundergoing a reaction to form one or more hydrocarbon crosslinkers. Insome embodiments, the polypeptide comprises any sequence selected fromTable 1 or variant thereof, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are eachan α,α-disubstituted amino acid. In some embodiments, the polypeptidedescribed herein comprises any sequence selected from Table 1, whereinXaa₁ and Xaa₂ are each an α,α-disubstituted amino acid and a hydrocarboncrosslinker is present between Xaa₃ and Xaa₄. In some embodiments, thepolypeptide described herein comprises any sequence selected from Table1, wherein Xaa₃ and Xaa₄ are each an α,α-disubstituted amino acid and ahydrocarbon crosslinker is present between Xaa₁ and Xaa₂. In someembodiments, the α,α-disubstituted amino acid is an α-methyl, α-alkenylamino acid. In some embodiments, the α-methyl, α-alkenyl amino acid isan α-methyl, α-alkenyl alanine. In some embodiments, the α-methyl,α-alkenyl alanine is selected from (S)-2-(4′-pentenyl)alanine,(R)-2-(4′-pentenyl)alanine, (S)-2-(7′-octenyl)alanine, and(R)-2-(7′-octenyl)alanine.

In a certain embodiment, the polypeptide or variant thereof describedherein is a stapled polypeptide comprising one or more hydrocarboncrosslinkers and comprises any sequence selected from Table 1 or variantthereof. In some embodiments, the polypeptide is a stapled polypeptidecomprising one hydrocarbon crosslinker, wherein Xaa₁ and Xaa₂ are eachalanine and the hydrocarbon crosslinker is present between Xaa₁ andXaa₂. In other embodiments, the polypeptide is a stapled polypeptidecomprising two hydrocarbon crosslinkers, wherein (1) Xaa₁, Xaa₂, Xaa₃,and Xaa₄ are each alanine, (2) a first hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂, and (3) a second hydrocarbon crosslinkeris present between Xaa₃ and Xaa₄. In some embodiments, the hydrocarboncrosslinker has an S-configuration on both ends. In some embodiments,the hydrocarbon crosslinker has an R-configuration on both ends. In someembodiments, the hydrocarbon crosslinker has an S-configuration on oneend and an R-configuration on the other end.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁QEQXaa₂E (SEQ ID NO: 35),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of SXaa₁EQLXaa₂H (SEQ ID NO: 36),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of QXaa₁QLEXaa₂R (SEQ ID NO: 37),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of EXaa₁LEHXaa₂E (SEQ ID NO: 38),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of QXaa₁EHRXaa₂R (SEQ ID NO: 39),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁HREXaa₂S (SEQ ID NO: 40),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of EXaa₁RERXaa₂L (SEQ ID NO: 41),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of HXaa₁ERSXaa₂Q (SEQ ID NO: 42),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁RSLXaa₂T (SEQ ID NO: 43),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of EXaa₁SLQXaa₂L (SEQ ID NO: 44),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁LQTXaa₂R (SEQ ID NO: 45),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of SXaa₁QTLXaa₂D (SEQ ID NO: 46),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁TLRXaa₂I (SEQ ID NO: 47),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of QXaa₁LRDXaa₂Q (SEQ ID NO: 48),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of TXaa₁RDIXaa₂R (SEQ ID NO: 49),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁DIQXaa₂B (SEQ ID NO: 50),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁IQRXaa₂L (SEQ ID NO: 51),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of DXaa₁QRBXaa₂F (SEQ ID NO: 52),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LRXaa₁IQRXaa₂L (SEQ ID NO: 53),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LQTLRXaa₁IQRXaa₂L (SEQ ID NO: 54),wherein Xaa₁ and Xaa₂ are each (S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 55), wherein Xaa₂, Xaa₃ and Xaa₄ are each (S)-2-(4′-pentenyl)alaninewhereas Xaa₁ is (R)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁DQXaa₂DRXaa₃DQRXaa₄DH (SEQ IDNO: 56), wherein Xaa₂, Xaa₃ and Xaa₄ are each (S)-2-(4′-pentenyl)alaninewhereas Xaa₁ is (R)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LEXaa₂LRXaa₃IERXaa₄L (SEQ IDNO: 57), wherein Xaa₂, Xaa₃ and Xaa₄ are each (S)-2-(4′-pentenyl)alaninewhereas Xaa₁ is (R)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 58), wherein Xaa₂, Xaa₃ and Xaa₄ are each (S)-2-(4′-pentenyl)alaninewhereas Xaa₁ is (R)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LQXaa₁LRDIQRXaa₂L (SEQ ID NO: 59),wherein Xaa₁ is (R)-2-(7′-octenyl)alanine and Xaa₂ is(S)-2-(4′-pentenyl)alanine.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁QEQXaa₂E (SEQ ID NO: 60),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of SXaa₁EQLXaa₂H (SEQ ID NO: 61),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of QXaa₁QLEXaa₂R (SEQ ID NO: 62),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH₂— CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of EXaa₁LEHXaa₂E (SEQ ID NO: 63),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of QXaa₁EHRXaa₂R (SEQ ID NO: 64),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁HREXaa₂S (SEQ ID NO: 65),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of EXaa₁RERXaa₂L (SEQ ID NO: 66),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of HXaa₁ERSXaa₂Q (SEQ ID NO: 67),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁RSLXaa₂T (SEQ ID NO: 68),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of EXaa₁SLQXaa₂L (SEQ ID NO: 69),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁LQTXaa₂R (SEQ ID NO: 70),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of SXaa₁QTLXaa₂D (SEQ ID NO: 71),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁TLRXaa₂I (SEQ ID NO: 72),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of QXaa₁LRDXaa₂Q (SEQ ID NO: 73),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of TXaa₁RDIXaa₂R (SEQ ID NO: 74),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LXaa₁DIQXaa₂B (SEQ ID NO: 75),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁IQRXaa₂L (SEQ ID NO: 76),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of DXaa₁QRBXaa₂F (SEQ ID NO: 77),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of LRXaa₁IQRXaa₂L (SEQ ID NO: 78),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide described herein comprises and/orconsists of an amino acid sequence of LQTLRXaa₁IQRXaa₂L (SEQ ID NO: 79),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration at both ends.

In some embodiments, a polypeptide the polypeptide described hereincomprises or consists of an amino acid sequence ofXaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ ID NO: 80), wherein (1) Xaa₃ and Xaa₄ areeach (S)-2-(4′-pentenyl)alanine, (2) Xaa₁ and Xaa₂ are alanine, and (3)a hydrocarbon crosslinker is present between Xaa₁ and Xaa₂. In theseembodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on one endand having an R-configuration on the other end.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 81), wherein (1) Xaa₁ is (R)-2-(4′-pentenyl)alanine and Xaa₂ is(S)-2-(4′-pentenyl)alanine, (2) Xaa₃ and Xaa₄ are alanine, and (3) ahydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 82), wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a firsthydrocarbon crosslinker is present between Xaa₁ and Xaa₂ and a secondhydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the first hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on one endand an R-configuration on the other end whereas the second hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁DQXaa₂DRXaa₃DQRXaa₄DH (SEQ IDNO: 83), wherein (1) Xaa₃ and Xaa₄ are each (S)-2-(4′-pentenyl)alanine,(2) Xaa₁ and Xaa₂ are alanine, and (3) a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on one end and having an R-configuration on the otherend.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁DQXaa₂DRXaa₃DQRXaa₄DH (SEQ IDNO: 84), wherein (1) Xaa₁ is (R)-2-(4′-pentenyl)alanine and Xaa₂ is(S)-2-(4′-pentenyl)alanine, (2) Xaa₃ and Xaa₄ are alanine, and (3) ahydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁DQXaa₂DRXaa₃DQRXaa₄DH (SEQ IDNO: 85), wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a firsthydrocarbon crosslinker is present between Xaa₁ and Xaa₂ and a secondhydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the first hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on one endand an R-configuration on the other end whereas the second hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LEXaa₂LRXaa₃IERXaa₄L (SEQ IDNO: 86), wherein (1) Xaa₃ and Xaa₄ are each (S)-2-(4′-pentenyl)alanine,(2) Xaa₁ and Xaa₂ are alanine, and (3) a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on one end and having an R-configuration on the otherend.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LEXaa₂LRXaa₃IERXaa₄L (SEQ IDNO: 87), wherein (1) Xaa₁ is (R)-2-(4′-pentenyl)alanine and Xaa₂ is(S)-2-(4′-pentenyl)alanine, (2) Xaa₃ and Xaa₄ are alanine, and (3) ahydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LEXaa₂LRXaa₃IERXaa₄L (SEQ IDNO: 88), wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a firsthydrocarbon crosslinker is present between Xaa₁ and Xaa₂ and a secondhydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the first hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on one endand an R-configuration on the other end whereas the second hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 89), wherein (1) Xaa₃ and Xaa₄ are each (S)-2-(4′-pentenyl)alanine,(2) Xaa₁ and Xaa₂ are alanine, and (3) a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on one end and having an R-configuration on the otherend.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 90), wherein (1) Xaa₁ is (R)-2-(4′-pentenyl)alanine and Xaa₂ is(S)-2-(4′-pentenyl)alanine, (2) Xaa₃ and Xaa₄ are alanine, and (3) ahydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on both ends.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 91), wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a firsthydrocarbon crosslinker is present between Xaa₁ and Xaa₂ and a secondhydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the first hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on one endand an R-configuration on the other end whereas the second hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on both ends.

In some embodiments, a polypeptide described herein comprises and/orconsists of an amino acid sequence of LQXaa₁LRDIQRXaa₂L (SEQ ID NO: 92),wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂. In these embodiments, the hydrocarboncrosslinker is —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—, having anR-configuration on one end and an S-configuration on the other end. Insome embodiments, the polypeptide or variant described herein furthercomprises a chemical modification at the N-terminus and/or C-terminus.In some embodiment, the N-terminus of the polypeptide or variantdescribed herein is further modified. In some embodiments, theN-terminus of the polypeptide or variant described herein is modified toan acetyl group. In some embodiments, the C-terminus of the polypeptideor variant described herein is further modified. In some embodiments,the C-terminus of the polypeptide or variant described herein ismodified with NH₂. In some embodiments, the C-terminus of thepolypeptide or variant described herein is modified with one, two, ormore units of β-alanine, 2-Naphthylalanine, and/or 2-Naphthylalanine,optionally linked to one, two, or more units of β-alanine, wherein thecarboxyl group of the C-terminus modification is optionally furthermodified with NH₂. In some embodiments, the N-terminus and/or theC-terminus of the polypeptide or variant described herein are furtherprotected by fluorenylmethyloxycarbonyl group (Fmoc). In someembodiments, the polypeptide or variant is further conjugated to one ormore chemical moieties that improve e.g., cell permeability, solubility,and stability of a polypeptide. In some embodiment, the polypeptide orvariant is conjugated to a TAT polypeptide (GRKKRRQRRRPQ (SEQ ID NO:93)).

In some embodiments, the polypeptide or variant consists of an aminoacid sequence selected from Table 1, wherein the N-terminus of thepolypeptide or variant is further modified with an acetyl group and theC-terminus of the polypeptide or variant is modified with NH₂.

In some embodiments, the polypeptide or variant consists of an aminoacid sequence of LRXaa₁IQRXaa₂L (SEQ ID NO: 94), wherein Xaa₁ and Xaa₂are each alanine and a hydrocarbon crosslinker is present between Xaa₁and Xaa₂. In these embodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration at both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified with2-Naphthylalanine linked to two units of β-alanine, wherein the carboxylgroup of the second β-alanine unit is modified with NH₂(2-Nal-β-Ala-β-Ala-NH₂). This polypeptide may be referred as “WX-021” inthis application.

In some embodiments, the polypeptide or variant consists of an aminoacid sequence of LRXaa₁IQRXaa₂L (SEQ ID NO: 95), wherein Xaa₁ and Xaa₂are each alanine and a hydrocarbon crosslinker is present between Xaa₁and Xaa₂. In these embodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration at both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified withtwo units of β-alanine, wherein the carboxyl group of the secondβ-alanine unit is modified with NH₂ (β-Ala-β-Ala-NH₂). This polypeptidemay be referred as “WX-022” in this application.

In some embodiments, the polypeptide or variant consists of an aminoacid sequence of LRXaa₁IQRXaa₂L (SEQ ID NO: 96), wherein Xaa₁ and Xaa₂are each alanine and a hydrocarbon crosslinker is present between Xaa₁and Xaa₂. In these embodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration at both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified with2-Naphthylalanine, wherein the carboxyl group of 2-Naphthylalanine ismodified with NH₂ (2-Nal-NH₂). This polypeptide may be referred as“WX-023” in this application.

In some embodiments, the polypeptide or variant consists of an aminoacid sequence of LQTLRXaa₁IQRXaa₂L (SEQ ID NO: 97), wherein Xaa₁ andXaa₂ are each alanine and a hydrocarbon crosslinker is present betweenXaa₁ and Xaa₂. In these embodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration at both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified with2-Naphthylalanine linked to two units of β-alanine. In theseembodiments, the polypeptide may be further conjugated to a TAT peptide(GRKKRRQRRRPQ (SEQ ID NO: 93)). This polypeptide may be referred as“WX-029” in this application.

In some embodiments, the polypeptide or variant consists of an aminoacid sequence of Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ ID NO: 98), wherein Xaa₁,Xaa₂, Xaa₃ and Xaa₄ are each alanine and a first hydrocarbon crosslinkeris present between Xaa₁ and Xaa₂ and a second hydrocarbon crosslinker ispresent between Xaa₃ and Xaa₄. In these embodiments, the firsthydrocarbon crosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on one end and an R-configuration on the other end,whereas the second hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified with2-Naphthylalanine linked to two units of β-alanine. In theseembodiments, the polypeptide may be further conjugated to a TAT peptide(GRKKRRQRRRPQ (SEQ ID NO: 93)). This polypeptide may be referred as“WX-036” in this instant application.

In some embodiments, a polypeptide described herein consists of an aminoacid sequence of Xaa₁DQXaa₂DRXaa₃DQRXaa₄DH (SEQ ID NO: 99), whereinXaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a first hydrocarboncrosslinker is present between Xaa₁ and Xaa₂ and a second hydrocarboncrosslinker is present between Xaa₃ and Xaa₄. In these embodiments, thefirst hydrocarbon crosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, havingan S-configuration on one end and an R-configuration on the other endwhereas the second hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified withtwo units of β-alanine, wherein the carboxyl group of the secondβ-alanine unit is modified with NH₂ (β-Ala-β-Ala-NH₂). This polypeptidemay be referred as “WX-037” in this application.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of Xaa₁LEXaa₂LRXaa₃IERXaa₄L (SEQ IDNO: 100), wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a firsthydrocarbon crosslinker is present between Xaa₁ and Xaa₂ and a secondhydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the first hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on one endand an R-configuration on the other end whereas the second hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on both ends. In these embodiments, the N-terminus ofthe polypeptide or variant may further be modified with an acetyl group.In these embodiments, the C-terminus of the polypeptide or variant maybe further modified with 2-Naphthylalanine linked to two units ofβ-alanine, wherein the carboxyl group of the second β-alanine unit ismodified with NH₂ (2-Nal-β-Ala-β-Ala-NH₂). This polypeptide may bereferred as “WX-038” in this application.

In some embodiments, a polypeptide described herein comprises orconsists of an amino acid sequence of RXaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ IDNO: 101), wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a firsthydrocarbon crosslinker is present between Xaa₁ and Xaa₂ and a secondhydrocarbon crosslinker is present between Xaa₃ and Xaa₄. In theseembodiments, the first hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on one endand an R-configuration on the other end whereas the second hydrocarboncrosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having anS-configuration on both ends. In these embodiments, the N-terminus ofthe polypeptide or variant may further be modified with an acetyl group.In these embodiments, the C-terminus of the polypeptide or variant maybe further modified with 2-Naphthylalanine linked to two units ofβ-alanine, wherein the carboxyl group of the second β-alanine unit ismodified with NH₂ (2-Nal-β-Ala-β-Ala-NH₂). This polypeptide may bereferred as “WX-039” in this application.

In some embodiments, a polypeptide described herein comprises and/orconsists of an amino acid sequence of LQXaa₁LRDIQRXaa₂L (SEQ ID NO:102), wherein Xaa₁ and Xaa₂ are each alanine and a hydrocarboncrosslinker is present between Xaa₁ and Xaa₂. In these embodiments, thehydrocarbon crosslinker is —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—,having an R-configuration on one end and an S-configuration on the otherend. In these embodiments, the N-terminus of the polypeptide or variantmay further be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified with2-Naphthylalanine linked to two units of β-alanine, wherein the carboxylgroup of the second β-alanine unit is modified with NH₂(2-Nal-β-Ala-β-Ala-NH₂). This polypeptide may be referred as “WX-040” inthis application.

In some embodiments, the polypeptide or variant consists of an aminoacid sequence of LQTLRXaa₁IQRXaa₂L (SEQ ID NO: 103), wherein Xaa₁ andXaa₂ are each alanine and a hydrocarbon crosslinker is present betweenXaa₁ and Xaa₂. In these embodiments, the hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration at both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified with2-Naphthylalanine, wherein the carboxyl group of 2-Naphthylalanine ismodified with NH₂ (2-Nal-NH₂). This polypeptide may be referred as“WX-024” in this application. The chemical structure of WX-024 is shownin FIG. 1A.

In some embodiments, the polypeptide or variant consists of an aminoacid sequence of Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ ID NO: 104), whereinXaa₁, Xaa₂, Xaa₃ and Xaa₄ are each alanine and a first hydrocarboncrosslinker is present between Xaa₁ and Xaa₂ and a second hydrocarboncrosslinker is present between Xaa₃ and Xaa₄. In these embodiments, thefirst hydrocarbon crosslinker is —CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, havingan S-configuration on one end and an R-configuration on the other end,whereas the second hydrocarbon crosslinker is—CH2-CH2-CH2-CH═CH—CH2-CH2-CH2-, having an S-configuration on both ends.In these embodiments, the N-terminus of the polypeptide or variant mayfurther be modified with an acetyl group. In these embodiments, theC-terminus of the polypeptide or variant may be further modified with2-Naphthylalanine linked to two units of β-alanine, wherein the carboxylgroup of the second β-alanine unit is modified with NH₂(2-Nal-β-Ala-β-Ala-NH₂). This polypeptide may be referred as “WX-035” inthis instant application. The chemical structure of WX-035 is shown inFIG. 1B.

3. Hydrocarbon Crosslinkers

The polypeptides described herein can encompass a stabilized polypeptidehaving one or more hydrocarbon crosslinkers. The hydrocarbon crosslinkermay confer a structural constraint(s) of the α-helix of the polypeptidederived from the HD2 domain of BCL9. In one embodiment, the α-helix of apolypeptide derived from the HD2 domain of the BCL9 peptide isstabilized by one or more hydrocarbon crosslinkers. As used herein, theterms “hydrocarbon crosslinker” and “crosslinker” (also known as ahydrocarbon staple, hydrocarbon linker, or a metathesized crosslinker)are used interchangeably and refer to a chemical linker between twoamino acids, in which the linker significantly enhances and/orreinforces the secondary structure of a given polypeptide. Thehydrocarbon crosslinker as described herein may be based on theincorporation of natural or non-natural amino acids that restrict thestructural flexibility of the polypeptide compared to a wildtype (i.e.non-crosslinked) peptide.

For instance, the hydrocarbon crosslinker as used herein may enhance thestability of the α-helical structure of a polypeptide derived from theHD2 domain of human BCL9. The hydrocarbon crosslinker may extend acrossthe length of one or more α-helical turns. As it is generally understoodthat one α-helical turn comprise about 3 to 4 amino acids, it will alsobe appreciated that amino acids positioned at e.g., i and i+3; i andi+4; or i and i+7 would be ideal positions for introducing a hydrocarboncrosslinker. For example, a polypeptide having a sequence ofX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ may be crosslinked by a hydrocarbon crosslinkere.g., between X₁ and X₄, between X₁ and X₅, or between X₁ and X₈.However, other positions may also be considered with alternativeintervals to enhance the stability of α-helical structure or other knownstructures (e.g., β-sheet structure) and/or to introduce a variation tothe secondary structure by e.g., altering the number of amino acids perhelical turn. Thus, any of the polypeptides derived from the HD2 domainof human BCL9 protein, as discussed herein, may be crosslinked at anysuitable positions.

In some embodiments, a stabilized polypeptide described herein comprisesa hydrocarbon crosslink at positions i and i+3, i and i+4, or i and i+7.In certain embodiments, the crosslink links a first amino acid (i) and asecond amino acid (i+3) that occurs 3 amino acids downstream for thefirst amino acid. In certain embodiments, the crosslink links a firstamino acid (i) and a second amino acid (i+4) that occurs 4 amino acidsdownstream from the first amino acid. In certain embodiments, thecrosslink links a first amino acid (i) and a second amino acid (i+7)that occurs 7 amino acids downstream from the first amino acid.

In some embodiments, a polypeptide described herein comprises two ormore hydrocarbon crosslinkers. In some embodiments, the peptide isstabilized with two hydrocarbon crosslinkers. In some embodiments, thepeptide is stabilized with three crosslinkers. For example, threecrosslinkers may be used for longer structures, such as those with 24amino acids. The use of two or more hydrocarbon crosslinkers could bebeneficial for enhancing and/or reinforcing the secondary structure of agiven polypeptide as compared to a polypeptide comprising no hydrocarboncrosslinker or one hydrocarbon crosslinker. Multiple hydrocarboncrosslinks within a single peptide may confer additional stability tothe alpha helix, such as better stability and an improvedpharmacokinetic profile. In some embodiments, multiple crosslinks arepresent within a single polypeptide. In certain embodiments, thesecrosslinks may be sequential with the same distance between amino acidsof the crosslink. In one embodiment, two crosslinks are present atpositions i and i+3 and positions i′ and i′+3. The position i′ mayreside between i and i+3 or reside after the position i+3. In oneembodiment, two crosslinks are at positions i and i+4 and positions i′and i′+4 positions. The position i′ may reside between i and i+4 orreside after the position i+4. In one embodiment, two crosslinks are atpositions i and i+7 positions and positions i′ and i′+7. The position i′may reside between i and i+7 or reside after the position i+7.

In certain embodiments, two crosslinks within a single peptide are atmixed positions, wherein one crosslink is at position i and i+3, i andi+4, or i and i+7; and the other crosslink has a different spacingbetween the amino acids that are linked. In some embodiments, thepolypeptide described herein has two hydrocarbon crosslinkers whereinone crosslinker is at position i and i+3 and the other crosslinker atposition i′ and i′+4. In some embodiments, the position i′ may residebetween i and i+3 or reside after the position i+3. In some embodiments,the position i may reside between i′ and i′+4 or reside after theposition i′+4.

Various hydrocarbon crosslinkers are known in the art. (Azzarito et al.,Nature Chemistry 5: 161-173 (2013)). In some embodiments, a hydrocarboncrosslinker disclosed herein is generated by connecting twoα,α-disubstituted amino acids incorporated into a single polypeptide. Insome embodiments, the hydrocarbon crosslinker is generated by a ringclosing metathesis reaction connecting two α,α-disubstituted aminoacids. A ring closing metathesis (also referred as a ring closing olefinmetathesis) is known in the art (Kim et al., Nature Protocols 6: 761-771(2011)).

The length of a hydrocarbon crosslinker as described herein may varydepending on the length of the substituents of the α,α-disubstitutedamino acid. For instance, by using a suitable α,α-disubstituted aminoacid, a hydrocarbon crosslinker generated by a ring closing metathesisreaction may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 carbons. In some embodiments, the hydrocarbon crosslinker hasa length of 8-12 carbons. In some embodiments, the hydrocarboncrosslinker has a length of 8 or 11 carbons. In certain embodiments, thehydrocarbon linker is 8-carbons in length. In certain embodiments, thehydrocarbon linker is 11-carbons in length. The length of a hydrocarboncrosslinker may be adjusted depending on whether the hydrocarboncrosslinker would extend over one, two, three, or more helical turns.For instance, a hydrocarbon crosslinker having a length of 8 carbons maybe used for connecting i and i+3 or i and i+4 positions. A hydrocarboncrosslinker having a length of 11 carbons may be used to connect i andi+7 positions.

In some embodiments, the hydrocarbon crosslinker is an alkenylcrosslinker. In some embodiments, a hydrocarbon crosslinker describedherein is generated by connecting two α-alkyl, α-alkenyl amino acids. Insome embodiments, the α-alkyl, α-alkenyl amino acid is an α-methyl,α-alkenyl amino acid. In some embodiments, the α-methyl, α-alkenyl aminoacid is an α-methyl, α-alkenyl alanine. In some embodiments, theα-methyl, α-alkenyl alanine is selected from (S)-2-(4′-pentenyl)alanine,(R)-2-(4′-pentenyl)alanine, (S)-2-(7′-octenyl)alanine, and(R)-2-(7′-octenyl)alanine. In some embodiments, the hydrocarboncrosslinker is —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—. In some embodiments, thehydrocarbon crosslinker is —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—.

In some embodiments, a hydrocarbon crosslinker described herein may havechirality at either end of the crosslinker. In some embodiments, thehydrocarbon cross linker has an S-configuration on both ends. In someembodiments, the hydrocarbon cross linker has an R-configuration on bothends. In some embodiments, the hydrocarbon cross linker has anS-configuration on one end and an-R configuration on the other end. Insome embodiments, the hydrocarbon crosslinker is—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—, having an S-configuration on both ends.In some embodiments, the hydrocarbon crosslinker is—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—, having an R-configuration on both ends.In some embodiments, the hydrocarbon crosslinker is—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—, having an S-configuration on one endand an R-configuration on the other end. In some embodiments, thehydrocarbon crosslinker is —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—,having an S-configuration on both ends. In some embodiments, thehydrocarbon crosslinker is —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—,having an R-configuration on both ends. In some embodiments, thehydrocarbon crosslinker is —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—,having an S-configuration on one end and an R-configuration on the otherend.

A variety of non-hydrocarbon linkers are known in the literature (seeAzzarito et al., Nat Chem 5:161-173 (2013)). In some embodiments, thepeptide is stabilized by a crosslinker that is a disulfide bridge,lactam bridge, hydrogen-bonding surrogate, or triazole staple.

4. Preparation and Purification of Peptides with HydrocarbonCross-Linkers

The present disclosures also provide methods to manufacture apolypeptide described herein. Such methods may comprise generating apolypeptide that is capable of undergoing a reaction to form one or morehydrocarbon crosslinkers, wherein the polypeptide comprises any sequenceselected from Table 1 or a variant thereof. In some embodiments, themethod comprises generating a polypeptide comprising LQTLRXaa₁IQRXaa₂L(SEQ ID NO: 1), Xaa₁LQXaa₂LRXaa₃IQRXaa₄L (SEQ ID NO: 2), or a variantthereof. In some embodiments, Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are each anα,α-disubstituted amino acid; Xaa₁ and Xaa₂ are each anα,α-disubstituted amino acid and a hydrocarbon crosslinker is presentbetween Xaa₃ and Xaa₄; or Xaa₃ and Xaa₄ are each an α,α-disubstitutedamino acid and a hydrocarbon crosslinker is present between Xaa₁ andXaa₂. In some embodiments, the α,α-disubstituted amino acid is anα-methyl, α-alkenyl amino acid.

In some embodiments, a method for manufacturing a polypeptide describedherein comprises performing one or more chemical synthesis methods knownto the skilled artisan and described herein. See, for example, Fields etal., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W.H.Freeman & Co., New York, N.Y., 1992, p. 77; and Bird, G. H., et al.,Methods Enzymol 446, 369-86 (2008). In some embodiments, a method formanufacturing a polypeptide described herein comprises using solid phasesynthesis to generate the polypeptide. For instance, the polypeptidedescribed herein may be manufactured by the automated Merrifieldtechniques of solid phase synthesis with the alpha-NH₂ protected byeither t-Boc or Fmoc chemistry using side chain protected amino acidson, for example, an Applied Biosystems Peptide Synthesizer Model 430A or431 or the AAPPTEC multichannel synthesizer APEX 396.

In some embodiments, the polypeptide described herein may also bemanufactured in a high-throughput, combinatorial fashion, e.g., using ahigh-throughput multichannel combinatorial synthesizer. Other methods ofsynthesizing peptides are known in the art.

The methods to manufacture the polypeptides described herein may furthercomprise forming one or more hydrocarbon crosslinkers. The one or morehydrocarbon crosslinkers may be formed by subjecting the polypeptidedescribed herein to metal-mediated ring-closing olefin metathesis. Forinstance, the synthetic strategy for generating hydrocarbon crosslinkersbased on modified Ala residues (α-methyl, α-alkenyl amino acids) wouldbe known to those of ordinary skill in the art. The hydrocarboncrosslinker connects adjacent turns of the α-helix, flanked on each endby an α-methyl group. The basis of the chemistry to generate thishydrocarbon crosslinker is incorporation of two α-methyl, α-alkenylamino acids during synthesis of a peptide (see Kim 2011). A hydrocarboncrosslinker between these modified amino acids is then generated by useof a ruthenium-mediated ring-closing olefin metathesis. Following thering closure, the polypeptide may be deprotected and released from suchreaction, resulting in a polypeptide comprising one or more hydrocarboncrosslinkers.

The present disclosures also encompass methods of purifying apolypeptide manufactured according to the methods described herein. Insome embodiments, the polypeptide is purified by high-performance liquidchromatograph (HPLC). In some embodiments, the purified polypeptide issubstantially free of metal. As used herein, the term “substantiallyfree of metal” refers to a composition comprising a polypeptidedescribed herein and a metal at a concentration of less than about 0.5,1, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ppm. In someembodiments, the purified polypeptide is substantially free of metal,comprising less than about 0.5 ppm. In some embodiments, the purifiedpolypeptide comprises less than about 5 ppm. In some embodiments, thepurified polypeptide comprises less than about 20 ppm.

A polypeptide manufactured according to the method disclosed herein mayexist in various forms in the presence or absence of a solvent. Forinstance, the polypeptide may exist as powder, salt, liquid, crystal, orlyophilized composition. In some embodiments, the polypeptide describedherein exists as a salt form or crystal. Examples of suitable salt formsinclude acetate, trifluoroacetate, adipate, benzoate, benzenesulfonate,butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate,glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate,picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate,tosylate and undecanoate. The terms “crystal” and “crystallized” referto a polypeptide that exists in the form of a crystal. Crystals are oneform of the solid state of matter, which is distinct from other formssuch as the amorphous solid state or the liquid crystalline state. Thesalt form or crystal of a polypeptide described herein retains one ormore biological functions of the polypeptide that is manufactured in adifferent form (e.g., a powder form). In some embodiments, the salt formis trifluoroacetic salt, acetic salt, or hydrochloric salt. In someembodiments, the polypeptide described herein exists in a salt form andis stable at room temperature for at least one month. In someembodiment, the polypeptide described herein is stable at 2-8° C. for atleast one month.

A polypeptide described herein may be conjugated to one or more chemicalmoieties that are known to improve e.g., cell permeability, solubility,and stability of a given polypeptide. The term “conjugate” refers to apolypeptide that is chemically linked to a second chemical moiety. Insome embodiments, the polypeptide is conjugated to one or moremodifications e.g., a cell-permeability increasing moiety and a celltargeting moiety. Various modifications in these categories are known inthe art. For instance, the polypeptide described herein may bePEGylated, a modification known to facilitate cellular uptake, increasesbioavailability, increases blood circulation and half-life, alterspharmacokinetics, decreases immunogenicity and/or decreases the neededfrequency of administration. In some embodiments, the polypeptidedescribed herein is conjugated to Ant8 (Antennapedia 8-mer peptide), TATpeptide (GRKKRRQRRRPQ (SEQ ID NO: 93)), or one or more units ofβ-alanine.

In various embodiments, a polypeptide described herein may also beconjugated to an agent. In some embodiment, the agent is animmunoadhesion molecule, an imaging agent, a therapeutic agent, or acytotoxic agent. In an embodiment, the imaging agent is a radiolabel, anenzyme, a fluorescent label, a luminescent label, a bioluminescentlabel, a magnetic label, or biotin. In another embodiment, theradiolabel is ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho,or ¹⁵³Sm. In yet another embodiment, the therapeutic or cytotoxic agentis an anti-metabolite, an alkylating agent, an antibiotic, a growthfactor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, toxin, or an apoptotic agent, anti-cancer agent, animmunosuppressive agent, or an immune reaction stimulatory agent.

In various embodiments, an isolated nucleic acid encoding a polypeptidedisclosed herein is also provided. Also provided is a vector (e.g., anexpression vector) comprising the isolated nucleic acid disclosedherein.

In another aspect, a host cell is transformed with the vector disclosedherein. In an embodiment, the host cell is a prokaryotic cell, forexample, E. coli. In another embodiment, the host cell is a eukaryoticcell, for example, a protist cell, an animal cell, a plant cell, or afungal cell. In an embodiment, the host cell is a mammalian cellincluding, but not limited to, CHO, COS, NS0, SP2, PER.C6, or a fungalcell, such as Saccharomyces cerevisiae, or an insect cell, such as Sf9.

In various embodiments, a polypeptide disclosed herein can be preparedby culturing any one of the host cells disclosed herein in a culturemedium under conditions sufficient to produce the polypeptide.

5. Pharmaceutical Composition

In various embodiments, pharmaceutical compositions comprising one ormore of the polypeptides disclosed herein, either alone or incombination with other prophylactic agents, therapeutic agents, and/orpharmaceutically acceptable carriers, are provided. In some embodiments,the pharmaceutical composition may comprise one, two, three, or morepolypeptides described herein. The pharmaceutical compositionscomprising polypeptides provided herein are for use in, but not limitedto, diagnosing, detecting, or monitoring a disorder, in preventing,treating, managing, or ameliorating a disorder or one or more symptomsthereof, and/or in research.

A “pharmaceutically acceptable carrier” refers to e.g., any and allsolvents, solids, semisolids, liquid fillers, diluents, encapsulatingmaterials, formulation auxiliaries, media, isotonic and absorptiondelaying agents, for use with a polypeptide described herein tocomprises a “pharmaceutical composition” suitable for administration toa subject. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Supplementary active compounds canalso be incorporated into the compositions. The pharmaceuticallyacceptable carrier may be selected based on the use and/oradministration route of the composition.

The pharmaceutical compositions may be formulated into any of manypossible dosage forms, such as, e.g., tablets, capsules, gel capsules,powders, or granules. The pharmaceutical compositions may also beformulated as solutions, suspensions, emulsions, or mixed media. In someembodiments, the pharmaceutical compositions may be formulated aslyophilized formulations or aqueous solutions.

In some embodiments, a pharmaceutical composition may be formulated as asolution. For example, the polypeptides described herein may beadministered in an unbuffered solution, such as, e.g., in saline, inwater, or in dimethyl sulfoxide (DMSO). In some embodiments, thepolypeptides may also be administered in a suitable buffer solution. Forexample, the buffer solution may comprise acetate, citrate, prolamine,carbonate, or phosphate, or any combination thereof. In someembodiments, the buffer solution may be phosphate buffered saline (PBS).The pH and osmolality of the buffer solution containing the polypeptidescan be adjusted to be suitable for administering to a subject.

In some embodiments, the pharmaceutical compositions may be formulatedas suspensions in aqueous, non-aqueous, or mixed media. In someembodiments, the pharmaceutical composition is formulated in mixed mediacomprising water and DMSO. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers.

In some embodiments, the pharmaceutical composition is used for in vivoadministration and may be sterile. Sterility may be readilyaccomplished, e.g., by filtration through sterile filtration membranes.

In various embodiments, a pharmaceutical composition comprising apolypeptide described herein may further comprise at least oneadditional agent. In some embodiments, the at least one additional agentis selected from one or more of a checkpoint inhibitor, an EGFRinhibitor, a VEGF inhibitor, a VEGFR inhibitor, and an anti-cancer drug.

In some embodiments, the pharmaceutical composition described hereincomprises a checkpoint inhibitor. In an embodiment, the checkpointinhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or ananti-CTLA4 antibody. In an embodiment, the checkpoint inhibitor targetsa stimulatory checkpoint molecule such as e.g., CD27, CD40, OX40, GITR,or CD138. In yet another embodiment, the checkpoint inhibitor targets aninhibitory checkpoint molecule such as e.g., A2AR, B7-H3, B7-H4, B and Tlymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO),Killer-cell immunoglobulin-like receptor (KIR), Lymphocyte ActivationGene-3 (LAG3), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3),VISTA (C10orf54), or V-domain Ig suppressor of T cell activation.

In some embodiments, a pharmaceutical composition described hereincomprises an EGFR inhibitor. In an embodiment, the EGFR inhibitor iserlotinib, gefitinib, lapatinib, panitumumab, vandetanib, or cetuximab.

In some embodiments, a pharmaceutical composition described hereincomprises a VEGF or VEGFR inhibitor. In an embodiment, the VEGF or VEGFRinhibitor is pazopanib, bevacizumab, sorafenib, sunitinib, axitinib,ponatinib, regorafenib, vandetanib, cabozantinib, ramucirumab,lenvatinib, or ziv-aflibercept.

In some embodiments, a pharmaceutical composition described hereincomprises an anti-cancer drug. The anti-cancer drug may be selectedfrom: cyclophosphamide, methotrexate, 5-fluorouracil (5-FU),doxorubicin, mustine, vincristine, procarbazine, prednisolone,dacarbazine, bleomycin, etoposide, cisplatin, epirubicin, capecitabine,folinic acid, actinomycin, all-trans retinoic acid, azacitidine,azathioprine, bortezomib, carboplatin, chlorambucil, cytarabine,daunorubicin, docetaxel, doxifluridine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine,mercaptopurine, mitoxantrone, paclitaxel, pemetrexed, teniposide,tioguanine, topotecan, valrubicin, vinblastine, vindesine, vinorelbine,and oxaliplatin.

C. Biological Function of Polypeptides Derived from the HD2 Domain ofBCL9

The present disclosures encompass a polypeptide derived from the HD2domain of human BCL9 protein or a variant thereof, including stabilizedpeptides, wherein the polypeptide exhibits favorable biologicalfunctions in one or more of the following categories: (a) the bindingkinetics (on-rate, off-rate and affinity) of a polypeptide to BCL9, (b)potencies in various biochemical and cellular bioassays, (c) in vivoefficacies in relevant tumor models, (d) pharmacokinetic andpharmacodynamics properties, and (e) toxicological properties.

In some embodiments, a polypeptide described herein exhibits favorablebiological functions in some or each of the categories listed above,e.g., potencies in various biochemical and cellular bioassays includinga cell-based Wnt and/or β-catenin transcription assay. The term“biological function” refers the measured in vitro or in vivo action ofa polypeptide described herein. In some embodiments, the polypeptidedescribed herein has one or more improved biological functions ascompared to an unstapled wild-type human BCL9 HD2 domain or a wild-typefragment, selected from: (1) inhibiting binding of BCL9 to β-catenin;(2) inhibiting canonical Wnt/β-catenin signaling; (3) decreasingregulatory T cell survival; (4) decreasing expression of VEGF in atumor; (5) increasing CD4+ T cell and CD8+ T cell infiltration into atumor; (6) increasing T helper 17 (Th17) cell numbers in a tumor; (7)modulating dendritic cells in a tumor; (8) having a half-life (T½)greater than at least 2 hours when administrated to a subject; (9)inducing a tumor microenvironment favoring an immune reaction; and (10)inhibiting tumor growth, cancer stem cell proliferation, and/or tumormetastasis. Various assays are known in the art for measuring theseproperties and can be used herein.

In some embodiments, WX-024 has one or more improved biologicalfunctions as compared to another stapled peptide, an unstapled wild-typehuman BCL9 HD2 domain, or a wild-type fragment, wherein the biologicalfunction is selected from the biological functions described herein. Insome embodiments, the improved biological function is one or more ofactivity (e.g., as measured in vitro and/or in vivo) andpharmacokinetics. In some embodiments, WX-035 has one or more improvedbiological functions as compared to another stapled peptide, anunstapled wild-type human BCL9 HD2 domain, or a wild-type fragment,wherein the biological function is selected from the biologicalfunctions described herein. In some embodiments, the improved biologicalfunction is one or more of activity (e.g., as measured in vitro and/orin vivo) and pharmacokinetics.

The present disclosures also encompass a variant of the polypeptide,where the variant differs from the wild-type polypeptide in amino acidsequences and/or chemical structure, but retains one or more biologicalfunctions of the polypeptide.

As used herein, the terms “improve,” “increase,” “enhance,” “elevate,”“upregulate,” and “promote” one or more biological functions are allused interchangeably, and mean that the levels or activities of one ormore biological functions or readouts of the functions from in vitroand/or in vivo assays are increased above levels or activities observedin the absence of the polypeptide described herein and/or higher than avehicle or control polypeptide (e.g., an unstapled wild-type human BCL9HD2 domain, a polypeptide that does not comprise the core functionaldomain mediating the interaction between BCL9 and β-catenin, or acontrol polypeptide comprising a sequence not derived from the HD2domain of human BCL9 etc.). For instance, without being bound to anytheory, the polypeptide described herein may have an improved biologicalfunction in inhibiting binding of BCL9 to β-catenin, as assessed invarious in vitro assays e.g., in a homogenous time resolved fluorescence(HTRF) assay, as compared to a control polypeptide, e.g., an unstapledwild-type human BCL9 HD2 domain. In this context, a polypeptidedescribed herein may have an improved K_(D) value as compared to that ofthe control polypeptide or the polypeptide described herein may bind toβ-catenin in presence of the control polypeptide, indicating that thepolypeptide described herein has an improved ability to inhibit bindingof BCL9 to β-catenin as compared to the control polypeptide.

In some embodiments, the assay used to assess the biological function ofthe polypeptide described herein provides quantitative readout(s), andthe readouts observed with a disclosed polypeptide are at least 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more changed/improvedas compared to those observed with a vehicle control polypeptide (e.g.,an unstapled wild-type human BCL9 HD2 domain).

Various assays are known in the art for measuring biologicalactivity/function, several of which are described herein as non-limitingexamples described for illustrative purpose only.

1. Binding of BCL9 to β-Catenin

In some embodiments, a polypeptide or variant described herein inhibitsbinding of BCL9 to β-catenin in vitro and/or in vivo. In someembodiments, the polypeptide or variant disclosed herein inhibits theinteraction between Pygo and BCL9 or the formation of aPygo/BCL9/β-catenin complex. Pygopus (Pygo) and Legless (Lgs) werediscovered in Drosophila as new Wnt signaling components that areessential for Armadillo-mediated transcription during normal development(Belenkaya et al., Development (2002) 129(17): 4089-4101). Pygo andBCL9/Legless transduce the Wnt signal by promoting the transcriptionalactivity of beta-catenin/Armadillo in normal and malignant cells. Theability of a polypeptide to inhibit binding of BCL9 to β-catenin can beassessed in various assays known in the art. In some embodiments, thepolypeptide described herein inhibits binding of BCL9 to β-catenin whenassessed in a Homogeneous Time Resolved Fluorescence (HTRF) bindingassay. In this assay, a polypeptide is conjugated to a tag that canrecognize another tag attached to its target protein (i.e., β-catenin).When the polypeptide is bound to the target protein and therefore thetwo tags are in proximity, a signal is generated and can bequantitatively read to calculate the binding affinity of thepolypeptide. In some embodiments, the binding affinity of thepolypeptide in this assay is compared against that of a controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9) todetect improved binding affinity as compared to that of the controlpolypeptide, indicating that the polypeptide likely would inhibitbinding of BCL9 to β-catenin more efficiently than the controlpolypeptide. The assay may be conducted in the presence or absence of anuntagged control polypeptide. The assay may also be conducted by tagginga control polypeptide (e.g., an unstapled wild-type HD2 domain humanBCL9) in the presence or absence of an untagged polypeptide describedherein (e.g., WX-024).

In some embodiments, a polypeptide described herein inhibits binding ofBCL9 to β-catenin when assessed in an Amplified Luminescence ProximityHomogeneous Assay (ALPHA). In this assay, a polypeptide is conjugated toa donor bead and its target protein (i.e., β-catenin) is attached to anacceptor bead. When the two beads are in proximity due to the binding ofthe polypeptide to the target protein, a signal is generated and thebinding affinity of the polypeptide can be quantitatively calculated. Insome embodiments, the binding affinity of the polypeptide in this assayis compared against that of a vehicle or control polypeptide (e.g., anunstapled wild-type HD2 domain human BCL9) to detect improved bindingaffinity as compared to that of the vehicle or control polypeptide,indicating that the polypeptide likely would inhibit binding of BCL9 toβ-catenin more efficiently than the control polypeptide. The assay maybe conducted in the presence or absence of an unconjugated controlpolypeptide. The assay may also be conducted by conjugating the controlpolypeptide in the presence or absence of an unconjugated polypeptidedescribed herein (e.g., WX-024).

In various embodiments, a polypeptide described herein inhibits bindingof BLC9 to β-catenin when assessed in a Wnt transcription assay. In someembodiments, the Wnt transcription assay is a cell-based assay. In someembodiments, the cell-based Wnt transcription assay is a GeneBLAzer®beta-lactamase (bla) reporter assay. Various cell lines, transformedcell lines or primary cells derived from a healthy subject or subjectsuffering from a disease can be used in this assay. A cell line known tobe dependent on canonical Wnt/β-catenin signaling for its survival mayalso be used. In some embodiments, CellSensor™ LEF/TCF-bla HCT-116 cellsare used in this reporter assay. These cells contain a beta-lactamase(BLA) reporter gene under the control of a β-catenin/LEF/TCF responseelement stably integrated into HCT-116 cells. As the cellsconstitutively express beta-lactamase, adding a polypeptide thatinhibits binding of BCL9 to β-catenin in this assay leads to reducedproduction of beta-lactamase. The efficiency of the polypeptide insuppressing Wnt transcription can therefore be quantitively calculatedin this assay. In some embodiments, a polypeptide described hereinsuppresses the Wnt transcription as measured in a GeneBLAzer®beta-lactamase (bla) reporter assay, which is indicative of the abilityof the polypeptide to inhibit binding of BCL9 to β-catenin. In someembodiments, a polypeptide tested in this assay shows an improved IC₅₀in suppressing Wnt transcription as compared to that of a vehicle orcontrol polypeptide (e.g., an unstapled wild-type HD2 domain humanBCL9), indicating that the disclosed polypeptide likely inhibits bindingof BCL9 to β-catenin more efficiently than the vehicle or controlpolypeptide.

In some embodiments, a polypeptide described herein inhibits binding ofBLC9 to β-catenin when assessed in a cell-viability assay. In someembodiments, the cell-viability assay is a CellTiterGlo luminescentassay, wherein the viability of cells is quantitatively measured.Various cell lines, transformed cell lines or primary cells derived froma healthy subject or subject suffering from a disease can be used inthis assay. In some embodiments, a polypeptide described hereinsuppresses cell growth in this assay more efficiently than a vehicle orcontrol polypeptide (e.g., an unstapled wild-type HD2 domain humanBCL9), indicating that the disclosed polypeptide likely inhibits bindingof BCL9 to β-catenin more efficiently than the vehicle or controlpolypeptide.

2. Canonical Wnt/β-Catenin Signaling

In certain embodiments, the polypeptides described herein can inhibitcanonical Wnt/β-catenin signaling. Canonical Wnt/β-catenin signaling canbe assessed in various in vitro and/or in vivo assays. In someembodiments, the effect of the polypeptide described herein on canonicalWnt/β-catenin signaling is assessed in a cell-based Wnt transcriptionassay, e.g., a GeneBLAzer® beta-lactamase (bla) reporter assay. TheGeneBLAzer® beta-lactamase (bla) reporter assay measures the strength ofcanonical Wnt/β-catenin signaling by its ability to control theβ-catenin/LEF/TCF response element and therefore can be used to assesswhether a test agent can attenuate or increase the strength of canonicalWnt/β-catenin signaling control of its transcription targets. In someembodiments, a polypeptide described herein suppresses the Wnttranscription as measured in a GeneBLAzer® beta-lactamase (bla) reporterassay, indicating that the polypeptide can inhibit canonicalWnt/β-catenin signaling. In some embodiments, the polypeptide in thisassay shows an improved IC₅₀ in suppressing Wnt transcription ascompared to that of a vehicle or control polypeptide (e.g., an unstapledwild-type HD2 domain human BCL9), indicating that the polypeptidedescribed herein has an improved ability to inhibit canonicalWnt/β-catenin signaling as compared to the vehicle or controlpolypeptide.

The ability of a polypeptide described herein to inhibit canonicalWnt/β-catenin signaling may also be assessed by measuring the geneexpression and/or protein expression of target genes that aretranscriptionally controlled by canonical Wnt/β-catenin signaling. Theexpression of target genes may be assessed in transformed cellscontacted with a polypeptide described herein or a subject administeredwith such polypeptide. The target genes include e.g., c-myc, ccnd1,cd44, LGR5, VEGFA, AXIN2, and LEFT. The expression level of one or moretarget genes associated with canonical Wnt/β-catenin signaling may beanalyzed using known methods in the art, e.g., cell staining, flowcytometry, western-blotting, and/or real-time quantitative PCR (rt-qPCR)analysis. In some embodiments, a polypeptide described herein reducesthe expression of one or more target genes in a cell. In someembodiments, the polypeptide described herein reduces the expression ofone or more target gene more efficiently than a vehicle or controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9).

3. Regulatory T Cell Survival

In some embodiments, a polypeptide described herein decreases regulatoryT cell survival. In some embodiments, when administered to a subject,the polypeptide decreases regulatory T cell survival locally (e.g., in atumor) and/or systemically (e.g., in blood). In some embodiments, whenadministered to a subject, the polypeptide decreases regulatory T cellsurvival as compared to a control polypeptide. Various markers, e.g.,CD4, FOXP3, and CD25, are known to be expressed on regulatory T cells.The ability of a polypeptide disclosed herein to decrease regulatory Tcell survival may be assessed by counting the total number of regulatoryT cells present in blood and/or a specific tissue such as a tumor. Forinstance, samples obtained from a subject contacted with a polypeptidedescribed herein may be stained with antibodies that detect markersassociated with regulatory T cells. The samples may also be processedand labeled with antibodies that detect such markers and analyzed byflow cytometry. Gene and/or protein expression of such markers may bedetermined in a sample and analyzed by e.g., western-blotting and/orrt-qPCR.

In some embodiments, a polypeptide described herein reduces the numberof regulatory T cells in blood and/or a tumor when administered to asubject. In some embodiments, the polypeptide reduces the expression ofone or more markers associated with regulatory T cells in one or moresamples obtained from a subject administered with the polypeptide. Insome embodiments, the polypeptide further reduces the expression of theone or more markers as compared to a vehicle or control polypeptide(e.g., an unstapled wild-type HD2 domain human BCL9), when assessed invivo.

4. VEGF Expression in a Tumor

In certain embodiments, a polypeptide described herein decreases theexpression of VEGF in a tumor when administered to a subject bearing thetumor. Various assays to measure the gene expression and/or proteinexpression of VEGF in a tumor sample can be employed. For instance,after contacting the subject with the polypeptide, tumor cells may becollected and stained with an anti-VEGF antibody to detect VEGF protein.The cells may also be analyzed by e.g., rt-qPCR to determine the geneexpression of VEGF. Other assays that indicate the change of VEGFexpression can be employed. For instance, tumor samples from a subjectcontacted with a polypeptide described herein may be analyzed to detectvarious angiogenic markers controlled by VEGF. In some embodiments, apolypeptide described herein decreases the expression of VEGF moreeffectively than a vehicle or control polypeptide (e.g., an unstapledwild-type HD2 domain human BCL9).

5. CD4+ and/or CD8+ T Cell Infiltration into Tumor

In some embodiments, a polypeptide described herein increases CD4+ Tcell and/or CD8+ T cell infiltration into a tumor when administered to asubject bearing the tumor. The infiltration of CD4+ T cells and/or CD8+T cells into a tumor may be assessed by counting the total number ofCD4+ T cells and/or CD8+ T cells present in a tumor or a sample (e.g., abiopsy) from the tumor. In some embodiments, a polypeptide describedherein increases CD4+ T cell and/or CD8+ T cell infiltration into atumor when administered to a subject bearing the tumor more effectivelythan a vehicle or control polypeptide (e.g., an unstapled wild-type HD2domain human BCL9). Various markers, e.g., CD4 and CD45, are known to beexpressed on CD4+ T cells, also referred as helper T cells. Variousmarkers, e.g., CD8 and CD45, are known to be expressed on CD8+ T cells,also referred as cytotoxic T cells. The ability of the polypeptide toincrease CD4+ and/or CD8+ T cell infiltration into a tumor may beassessed in vivo, by administering the polypeptide to a subject having atumor. A tumor sample can be collected from the subject and stained withantibodies that detect markers associated with CD4+/CD8+ T cells. Thesamples may also be processed and labeled with, e.g., antibodies thatdetect such markers and analyzed by, e.g., flow cytometry. Gene and/orprotein expression of such markers may also be determined in a sampleand analyzed by e.g., western-blotting and/or rt-qPCR. In someembodiments, a polypeptide described herein increases the total amountof CD4+ T cells and/or CD8+ T cells in a tumor as compared to a vehicleor control polypeptide (e.g., an unstapled wild-type HD2 domain humanBCL9).

In some embodiments, a polypeptide described herein increases the totalnumber of CD4+ T cells and/or CD8+ T cells in blood when administered toa subject. The systemic increase of CD4+ T cells and/or CD8+ T cells mayindicate increased CD4+ T cell and/or CD8+ T cell infiltration into aspecific tissue as well, e.g., a tumor. In some embodiments, apolypeptide described herein increases the amount of circulating CD4+ Tcells and/or CD8+ T cells in vivo as compared to a vehicle or controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9).

6. T Helper 17 Cell Infiltration into Tumor

In some embodiments, a polypeptide described herein increases T helper17 cell infiltration into a tumor when administered to a subject bearingthe tumor. The infiltration of T helper 17 cells into a tumor may beassessed by counting the total number of T helper 17 cells present inthe tumor. In some embodiments, a polypeptide described herein increasesT helper 17 cell infiltration into a tumor when administered to asubject bearing the tumor as compared to a vehicle or controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9).Various markers, e.g., IL-17, are known to be expressed on T helper 17cells. The ability of the polypeptide to increase T helper 17 cellinfiltration into a tumor may be assessed in vivo, by administering thepolypeptide to a subject having a tumor. A tumor sample can be collectedfrom the subject and stained with, e.g., antibodies that detect markersassociated with T helper 17 cells. The samples may also be processed andlabeled with antibodies that detect such markers and analyzed by flowcytometry. Gene and/or protein expression of such markers may also bedetermined in a sample and analyzed by e.g., western-blotting and/orrt-qPCR. The samples may be analyzed to detect the amount of IL-17present in the samples.

In some embodiments, a polypeptide described herein increases the totalamount of T helper 17 cells in blood when administered to a subject. Thesystemic increase of T helper 17 cells may indicate increased T helper17 cells infiltration into a specific tissue, e.g., a tumor. Thesystemic increase of T helper 17 cells may be assessed by measuring theamount of IL-17 present in blood samples collected from a subject. Insome embodiments, the polypeptide increases the amount of circulating Thelper 17 cells in a subject as compared to a vehicle or controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9). Insome embodiments, a polypeptide described herein increases the amount ofcirculating IL-17 in a subject as compared to a control polypeptide.

7. Dendritic Cells in Tumor

In some embodiments, a polypeptide described herein modulate dendriticcells present in a tumor when administered to a subject bearing thetumor. The number of dendritic cells present in a tumor may be assessed,e.g., by staining the tumor with antibodies that recognize one or moremarkers associated with dendritic cells. In some embodiments, apolypeptide described herein decreases dendritic cells present in atumor when administered to a subject bearing the tumor more effectivelythan a vehicle or control polypeptide (e.g., an unstapled wild-type HD2domain human BCL9). In some embodiments, a polypeptide described hereinincreases dendritic cells present in a tumor when administered to asubject bearing the tumor more effectively than a vehicle or controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9).Various markers, e.g., CD11c, are known to be expressed on dendriticcells. The ability of the polypeptide to decrease dendritic cells in atumor may be assessed in vivo, by administering the polypeptide to asubject. A tumor sample can be collected from the subject and stainedwith antibodies that detect markers associated with dendritic cells. Thesamples may also be processed and labeled, e.g., with antibodies thatdetect such markers and analyzed by, e.g., flow cytometry. Gene and/orprotein expression of such markers and analyzed by e.g.,western-blotting and rt-qPCR.

In some embodiments, a polypeptide described herein decreases the totalamount of dendritic cells in blood when administered to a subject. Insome embodiments, a polypeptide described herein increases the totalamount of dendritic cells in blood when administered to a subject. Thesystemic decrease of dendritic cells may indicate that the amount ofdendritic cells in a specific tissue, e.g., a tumor, has also decreased.In some embodiments, a polypeptide described herein decreases the amountof circulating dendritic cells in a subject as compared to a vehicle orcontrol polypeptide (e.g., an unstapled wild-type HD2 domain humanBCL9). In some embodiments, a polypeptide described herein increases theamount of circulating dendritic cells in a subject as compared to avehicle or control polypeptide (e.g., an unstapled wild-type HD2 domainhuman BCL9).

8. Half-Life in a Subject

In various embodiments, a polypeptide described herein has one or moreimproved pharmacokinetic parameters as compared to a vehicle or controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9). Suchpharmacokinetic parameters may include e.g., a maximum observedconcentration (C_(max)), time to reach the maximum concentration(T_(max)), terminal half-life (T_(1/2)), total body clearance (CL),volume of distribution (V_(z)), area under the curve from the time ofdosing to the last measurable concentration (AUC_(0-t)), area under thecurve from the time of dosing extrapolated to infinity (AUC_(0-inf)),and bioavailability.

Methods for assessing pharmacokinetics of an agent are known in the art.For instance, blood samples from a subject administered with apolypeptide described herein may be obtained at 5 min, 1, 2, 4, 6, 8,12, and 24 hours post-dose. The concentration of the polypeptide in theblood samples can be analyzed by various analytical tools, e.g., LC/MS.Based on the concentration of the polypeptide at each time point,pharmacokinetic parameters are calculated. As used herein, the term“maximum observed concentration (C_(max))” refers to the maximum serumconcentration that a polypeptide achieves after administration. Relatedto the concept of C_(max), the time to reach the maximum concentration(T_(max)) is the time that it takes for a polypeptide to reach themaximum serum concentration. The terms “terminal half-life (T_(1/2))”and “half-life (T_(1/2))” are used interchangeably and refer to the timethat a polypeptide takes to lose half of its serum concentration. Totalbody clearance (CL) represents the volume of blood completely cleared ofa polypeptide per unit time. The term “volume of distribution (V_(z))”refers to a theoretically calculated volume that would be required tocontain the total amount of a polypeptide administered to a subject atthe same concentration observed in the blood. The term “bioavailability”refers to the degree and rate at which a drug is absorbed into a livingsystem or is made available at the site of physiological activity.Bioavailability can be a function of several of the previously describedproperties, including stability, solubility, immunogenicity andpharmacokinetics, and can be assessed using methods known to one skilledin the art.

In some embodiments, a polypeptide described herein has an improvedhalf-life in a subject as compared to a control polypeptide. In someembodiments, the polypeptide has a half-life greater than at least 0.5,1, 2, 3, 5, or 8 hours when administered to a subject, or any timeperiod in between. In some embodiments, the polypeptide described hereinhas a half-life greater than at least 2 hours when administered to asubject. Pharmacokinetic parameters of the polypeptide may be assessedin a mammal including e.g., a mouse, a rat, or a human. The parametersmay also be assessed using various administration routes, e.g.,intravenous, intraperitoneal, subcutaneous, and intramuscularadministration routes. In some embodiments, the pharmacokineticparameters of the polypeptide described herein are assessed in mice. Insome embodiments, the pharmacokinetic parameters of the polypeptidedescribed herein are assessed in mice administered with the polypeptidesubcutaneously. In some embodiments, the pharmacokinetic parameters ofthe polypeptide described herein are assessed in humans. In someembodiments, the pharmacokinetic parameters of the polypeptide describedherein are assessed in humans after subcutaneous administration.

9. Tumor Microenvironment Favoring Immune Reaction

In various embodiments, a polypeptide described herein induces a tumormicroenvironment favoring an immune reaction. In various embodiments, apolypeptide described herein induces a tumor microenvironment morefavorable to an immune reaction than a vehicle or control polypeptide(e.g., an unstapled wild-type HD2 domain human BCL9).

As used herein, the term “tumor microenvironment” means a cellularmicroenvironment in and/or around a tumor, including various cellsrecruited to the tumors, blood vessels, immune cells, signalingmolecules, and extracellular matrix. See e.g., Balkwill et al., J CellSci (2012) 125: 5591-5596. The polypeptide described herein may alterthe composition of immune cells and/or signaling molecules in and/oraround a tumor, thereby eliciting an immune reaction in themicroenvironment around the tumor.

Various parameters may be used to assess a tumor microenvironment. Forinstance, an increased ratio between cytotoxic T cells and regulatory Tcells in and/or around tumor tissues may indicate that a tumormicroenvironment favors an immune reaction. A decreased amount ofdendritic cells and/or regulatory T cells in and/or around tumor tissuemay also indicate that a tumor microenvironment favors an immunereaction. Other parameters include increased circulating T cells inperipheral blood and an increased ratio between T helper 17 cells andregulatory T cells in and/or around tumor tissues. These parameters mayindicate that a tumor microenvironment favors an immune reaction.

In some embodiments, a polypeptide described herein may increase theratio between the amount of cytotoxic T cells and the amount ofregulatory T cells in a tumor microenvironment. In some embodiments, theratio change caused by the polypeptide is greater than that caused by avehicle or control polypeptide (e.g., an unstapled wild-type HD2 domainhuman BCL9).

In some embodiments, a polypeptide described herein may increase theratio between T helper 17 cells and regulatory T cells in a tumormicroenvironment. In some embodiment, the ratio change caused by thepolypeptide is greater than that caused by a vehicle or controlpolypeptide (e.g., an unstapled wild-type HD2 domain human BCL9).

10. Tumor Growth, Cancer Stem Cell Proliferation, and/or TumorMetastasis

As Wnt signaling is a regulator of tumor growth, the efficacy oftreatments that affect the binding of BCL9 to β-catenin, such as thestabilized peptides of the HD2 domain of the BCL9 peptide describedherein, may be assessed in animal models.

The in vivo efficacy of stabilized BCL9 peptides may be assessed inmodels of human cancers using, e.g., BALB/c nude mice, since xenograftsof human cancer cells will grow into tumors in these mice. For example,subcutaneous inoculation with Colo320DM tumor cells, a commerciallyavailable cell line derived from human colon cancer tissue, can be usedto form a tumor in BALB/c nude mice. Additional in vivo models are alsoavailable to assess the in vivo efficacy of a polypeptide disclosedherein. For instance, Human DLD-1 colon cancer cells can be implantedinto nude mice to assess tumor growth. The CT26 syngeneic mouse model ofcolon cancer may also be used, as it allows for assessment of tumorgrowth in the background of an intact immune system. Other types ofcancer cells, e.g., B16 melanoma, 4T1 breast cancer, Renca renal cancer,and Lewis Lung Cell lung cancer cells, may also be used in these knownanimal models to assess the in vivo efficacy of the polypeptidedisclosed herein.

By administering a polypeptide described herein to one or more animalmodels, the effect of the polypeptide in decreasing tumor growth in vivomay be assessed. In some embodiments, the polypeptide inhibits tumorgrowth in vivo more effectively than a vehicle or control polypeptide(e.g., an unstapled wild-type HD2 domain human BCL9). In someembodiments, the tumor mass/volume of a subject administered with thepolypeptide described herein is at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 99% smaller than that of a subject administeredwith the control polypeptide. From animal data on treatment withstabilized BCL9 peptides, the ability of the peptide to inhibit Wntsignaling can be assessed by e.g., staining of tissue samples withmarkers of Wnt signaling. These downstream markers of Wnt signalinginclude e.g., Axin2 and CD44.

Orthotopic mouse models may be used to assess the effects of apolypeptide described herein on tumor metastasis. For instance, anorthotropic animal model may be injected with cells carrying luciferaseconstruct and then administered with its assigned treatment. Thepresence of the injected cells can be detected by administeringluciferin substrate to each treated animal. The intensity of thebioluminescent signal can be quantitatively measured and used as anindicator of cell growth. In some embodiments, a polypeptide describedherein suppresses tumor metastasis more effectively than the controlpolypeptide, when assessed in an orthotopic mouse model. In someembodiments, the polypeptide reduces tumor growth in an orthotopic mousemodel at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%as compared to a vehicle or control polypeptide (e.g., an unstapledwild-type HD2 domain human BCL9).

In some embodiments, the effects of a polypeptide described herein oncancer stem cell proliferation may be assessed by measuring biomarkersof various cancer stem cells. For instance, the expression level of CD44and/or LGR5 may indicate the amount of cancer stem cells present in asample. A tumor sample can be collected from a subject and stained withantibodies that detect markers associated with cancer stem cells. Thesamples may also be processed and labeled, e.g., with antibodies thatdetect such markers and analyzed by, e.g., flow cytometry. Gene and/orprotein expression of such markers can be detected and analyzed by e.g.,western-blotting and rt-qPCR. In some embodiments, the polypeptidedescribed herein reduces the expression level of CD44 and/or LGR5 in atumor when administered to a tumor bearing subject. In some embodiments,a polypeptide described herein more effectively reduces the expressionlevel of CD44 and/or LGR5 than that of a vehicle or control polypeptide(e.g., an unstapled wild-type HD2 domain of human BCL9 protein).

D. Methods of Treatment with Stabilized BCL9 Peptides

1. Diseases with Aberrant Wnt/β-Catenin Signaling

Aberrant Wnt/β-catenin signaling has been implicated in the malignanttransformation of normal cells into cancerous cells (see Thakur 2013).Activation of Wnt signaling and β-catenin nuclear localization has beenlinked to a tumor phenotype in multiple models.

The present disclosure encompasses compositions for use and methods ofusing the stapled polypeptides disclosed herein to inhibit binding ofBCL9 to β-catenin in a subject by administering the polypeptide or apharmaceutical composition comprising the polypeptide to the subject.The present disclosure also encompasses inhibiting canonicalWnt/β-catenin signaling in a subject by administering a polypeptide orpharmaceutical composition disclosed herein. The present disclosuresfurther encompass methods of treating a disease in a subject byadministering a polypeptide or pharmaceutical composition describedherein to the subject. The disease may be a cancer or other tumorousdisease associated with aberrant canonical Wnt/β-catenin signaling.

As used herein, “patient” and “subject” may interchangeably refer to ananimal, such as a mammal or a human, being treated or assessed for adisease, disorder, or condition, at risk of developing a disease,disorder, or condition, or having or suffering from a disease, disorder,or condition. In some embodiments, the subject is a mammal. In someembodiments, the subject is a human.

In some embodiments, such disease, disorder, or condition may be adisease associated with aberrant canonical Wnt/β-catenin signaling,and/or which could benefit from inhibiting canonical Wnt/β-cateninsignaling. In some embodiments, such disease, disorder, or condition isa cancer. In some embodiments, the cancer is a cancer where BCL9 and/orβ-catenin are highly expressed. In some embodiments, the cancer is acancer where BCL9 and β-catenin are co-localized in the nucleus of acancer cell. In some embodiments, the cancer is selected from: familialadenomatous polyposis (FAP), ocular cancer, rectal cancer, colon cancer,colorectal cancer, cervical cancer, prostate cancer, breast cancer,bladder cancer, oral cancer, benign and malignant tumors, stomachcancer, liver cancer, pancreatic cancer, lung cancer, corpus uteri,ovarian cancer, prostate cancer, testicular cancer, renal cancer,brain/CNS cancer, throat cancer, multiple myeloma, skin melanoma, acutelymphocytic leukemia, acute myelogenous leukemia, Ewing's Sarcoma,Kaposi's Sarcoma, basal cell carcinoma and squamous cell carcinoma,small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma,hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx cancer,esophageal cancer, larynx cancer, lymphoma, neurofibromatosis, tuberoussclerosis, hemangiomas, gastric cancer, ovarian cancer, hepatocellularcarcinoma, and lymphangiogenesis. In some embodiments, the cancer iscolorectal cancer. In some embodiments, the cancer is gastric cancer. Insome embodiments, the cancer is ovarian cancer. In some embodiments, thecancer is Hepatocellular carcinoma. In some embodiments, the cancer isbreast cancer. In some embodiments, the cancer is prostate cancer. Insome embodiments, the cancer is skin melanoma. In some embodiments, thecancer is lung cancer.

In some embodiments, any of the polypeptide or variant disclosed hereinor a pharmaceutical composition comprising such polypeptide can be usedto treat a disease, e.g., a cancer, listed above. In some embodiment,the polypeptide or variant is WX-024. In some embodiments, thepolypeptide or variant is WX-035.

In some embodiments, a tumor volume in a subject is reduced by more than10%, 20%, 30%, 40%, or 50% (or any percentage inbetween) afteradministration of one or more dosages of a polypeptide described hereinor a pharmaceutical composition comprising the polypeptide, as comparedto that of a subject treated with a vehicle or an unstapled peptide. Incertain embodiments, the reduction is achieved after 1 week, 2 weeks, 3weeks, or more of administration (or any time period in between). Insome embodiments, the tumor volume of a subject is reduced by more than50%, as compared to that of a subject treated with a vehicle orunstapled peptide, after 2 weeks of administration. A suitable dosageand/or formulation of a pharmaceutical composition for administration toa subject could be determined by one of skill in the art as thematerials and techniques necessary for the various methods ofadministration are available and known in the art. See e.g., Formulationand delivery of peptides and proteins, ‘edition, Washington, ACS, pp.22-45 and Peptide and protein drug delivery, I’ edition, New York,Marcel Dekker, Inc., pp. 247-301. In some embodiment, the tumor volumeof a subject administered with WX-024 or a pharmaceutical compositioncomprising the polypeptide is reduced by more than 50%, as compared tothat of a subject treated with a vehicle or unstapled polypeptide, after2 weeks of administration. In some embodiment, the tumor volume of asubject administered with WX-035 or a pharmaceutical compositioncomprising the polypeptide is reduced by more than 10%-50%, as comparedto that of a subject treated with a vehicle or unstapled polypeptide,after 2 weeks of administration.

In various embodiments, the term “treatment” includes treatment of asubject (e.g. a mammal, such as a human) or a cell to alter the currentcourse of the subject or cell. Treatment includes the alteration of oneor more disease parameter, e.g., reduction in tumor volume or any otheroncologic measurement known to one of skill in the art. Treatmentincludes, e.g., administration of a polypeptide described herein or apharmaceutical composition comprising such polypeptide, and may beperformed either prophylactically or subsequent to the initiation of apathologic event or contact with an etiologic agent. Also included are“prophylactic” treatments, which can be directed to reducing the rate ofprogression of the disease or condition being treated, delaying theonset of that disease or condition, or reducing the severity of itsonset. “Treatment” or “prophylaxis” does not necessarily indicatecomplete eradication, cure, or prevention of the disease or condition orthe associated symptoms. In various embodiments, the term “treatment”may include relieving, slowing, or reversing the pathological processesor symptoms in a subject suffering from a cancer. In some embodiments,the term “treatment” may include decreasing tumor burden in a subjectsuffering from a cancer. In some embodiments, the term “treatment” mayinclude improving at least one symptom or measurable parameter of acancer. It will be apparent to one of skill in the art which biologicaland/or physiological parameters can be used to access the pathologicalprocess of the malignant disease.

Treatment, and the measured parameters of treatment, can be assessedafter administration of the polypeptide or pharmaceutical compositionalone or in combination with one or more additional therapeutic agents,e.g., as a single bolus or separate sequential administrations. Theadditional agent may be any of the additional therapeutic agentsmentioned herein or known to the skilled artisan. The polypeptide orpharmaceutical composition comprising the polypeptide, and/or theadditional agent, may be administered once or multiple times, dependingon the chosen regimen.

The present disclosures also encompass a polypeptide or pharmaceuticalcomposition disclosed herein for use in treating a disease in a subject.In some embodiment, the disease may benefit from suppressing canonicalWnt/β-catenin signaling. In some embodiments, the disease is a cancer.

The present disclosures further encompass uses of a polypeptide orpharmaceutical composition disclosed herein in the manufacture of amedicament for treating a disease in a subject. In some embodiment, thedisease may benefit from suppressing canonical Wnt/β-catenin signaling.In some an embodiment, the disease is a cancer.

In another embodiment, the disease treated is not cancer. In certainembodiments, the disease is a bone density defect, vascular defect ofthe eye, familial exudative vitreoretinopathy, early coronary disease,Alzheimer's disease, autosomal-dominant oligodontia, retinalangiogenesis, osteogenesis imperfecta, Tetra-Amelia syndrome,Mullerian-duct regression and virilization, SERKAL syndrome, Type IIdiabetes, Fuhrmann syndrome, odonto-onycho-dermal dysplasia, obesity,split hand/foot malformation, caudal duplication, tooth agenesis,skeletal dysplasia, focal dermal hypoplasia, autosomal recessiveanonychia, neural tube defects, or sclerosteosis and Van Buchem disease.

2. Administration of Stabilized BCL9 Peptides

As non-crosslinked (i.e. wildtype) peptides are normally metabolizedvery rapidly in vivo, peptide stabilization may improve thepharmacokinetic profile of these peptides, when administered to asubject. As used herein, the terms “administering,” or “administer”include delivery of the polypeptide described herein to a subject eitherby local or systemic administration. Administration may be by topical(including ophthalmic and to mucous membranes including vaginal andrectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer, intratracheal, intranasal),epidermal, transdermal, oral, or parenteral. Parenteral administrationincludes intravenous, subcutaneous, intraperitoneal, or intramuscularinjection or infusion; or intracranial, e.g., intrathecal orintraventricular, administration. Combinations of administration routesare also contemplated. The stapled peptide may be administered in atherapeutically-effective amount.

In some embodiments, a polypeptide or pharmaceutical compositiondescribed herein is administered intravenously. In some embodiments, apolypeptide or pharmaceutical composition described herein isadministered intraperitoneally. In some embodiments, a polypeptide orpharmaceutical composition described herein is administered daily,weekly, monthly, or any suitable interval that can be used for treatinga disease in a subject.

In some embodiments, administration of the stabilized peptide inhibitsWnt signaling in a subject. In some embodiments, administering of astabilized BCL9 peptide inhibits binding of BCL9 to β-catenin. In someembodiments, administering administration of a stabilized BCL9 peptideinhibits canonical Wnt/β-catenin signaling. In some embodiments,administering administration of a stabilized BCL9 peptide treat adisease in a subject.

3. Combination Therapy with Stabilized BCL9 Peptides

In certain embodiments, a polypeptide or pharmaceutical compositiondisclosed herein is administered with at least one additional agent. Insome embodiments, the at least one additional agent is selected from acheckpoint inhibitor, an EGFR inhibitor, a VEGF inhibitor, a VEGFRinhibitor, an anti-cancer drug. The additional agent may be administeredin the same pharmaceutical composition as the stapled peptide, or theymay be administered sequentially. The stapled peptide and the additionalagent may be administered in a therapeutically-effective amount.

In certain embodiments, the additional agent is a checkpoint inhibitor.Checkpoint inhibitors, such as checkpoint blocking antibodies, blocknormal negative regulators of T cell immunity, thereby increasing theimmune system's ability to control cancer in some patients (see Kyi andPostow, FEBS Letters 588: 368-376 (2013)). In certain embodiments, thecheckpoint inhibitor administered with a polypeptide or pharmaceuticalcomposition described herein inhibits PD1, PDL-1, and/or CTLA-4. In anembodiment, the checkpoint inhibitor is an anti-PD-1 antibody, ananti-PD-L1 antibody, and/or an anti-CTLA4 antibody. In certainembodiments, the checkpoint inhibitor is a checkpoint blocking antibodye.g., ipilimumab, nivolumab, or MK-3475. In an embodiment, thecheckpoint inhibitor targets a stimulatory checkpoint molecule such ase.g., CD27, CD40, OX40, GITR, or CD138. In yet another embodiment, thecheckpoint inhibitor targets an inhibitory checkpoint molecule such as,e.g., A2AR, B7-H3, B7-H4, B and T lymphocyte attenuator (BTLA),indoleamine 2,3-dioxygenase (IDO), Killer-cell immunoglobulin-likereceptor (KIR), Lymphocyte Activation Gene-3 (LAG3), T-cellImmunoglobulin domain and Mucin domain 3 (TIM-3), VISTA (C10orf54), orV-domain Ig suppressor of T cell activation.

Epidermal growth factor receptor (EGFR) inhibitors have shown efficacyin non-small cell lung cancer, as well as other types of cancer,especially in patients with genetic mutations in the EGFR or ALK(anaplastic lymphoma receptor tyrosine kinase) genes. In certainembodiments, the additional therapeutic agent administered with apolypeptide or pharmaceutical composition described herein is an EGFRinhibitor. In certain embodiments, the EGFR inhibitor is erlotinib,gefitinib, lapatinib, panitumumab, vandetanib, or cetuximab. In certainembodiments, a polypeptide or pharmaceutical composition describedherein is administered together with an EGFR inhibitor in patients whohave been determined to have molecular abnormalities in the EGFR or ALKgenes.

In an embodiment, a VEGF and/or VEGFR inhibitor is administered as thesecond agent. In some embodiments, the VEGF and/or VEGFR inhibitor isone or more of pazopanib, bevacizumab, sorafenib, sunitinib, axitinib,ponatinib, regorafenib, vandetanib, cabozantinib, ramucirumab,lenvatinib, and ziv-aflibercept.

In some embodiments, an anti-cancer drug is administered as the secondagent. In some embodiments, the anti-cancer drug is selected from one ormore of: cyclophosphamide, methotrexate, 5-fluorouracil (5-FU),doxorubicin, mustine, vincristine, procarbazine, prednisolone,dacarbazine, bleomycin, etoposide, cisplatin, epirubicin, capecitabine,folinic acid, actinomycin, all-trans retinoic acid, azacitidine,azathioprine, bortezomib, carboplatin, chlorambucil, cytarabine,daunorubicin, docetaxel, doxifluridine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine,mercaptopurine, mitoxantrone, paclitaxel, pemetrexed, teniposide,tioguanine, topotecan, valrubicin, vinblastine, vindesine, vinorelbine,and oxaliplatin.

In certain embodiments, a subject administered with a polypeptide orpharmaceutical composition disclosed herein is also treated withradiation therapy and/or chemotherapy before, after, or at the same timeas the polypeptide or pharmaceutical composition administration. Furthercombination therapies with the additional agents disclosed herein arealso contemplated.

4. Biomarkers

The present disclosures also encompass methods of measuring at least onebiomarker to monitor treatment efficacy of a polypeptide orpharmaceutical composition described herein or to select a subject fortreatment with such polypeptide or pharmaceutical composition. In someembodiments, the biomarker is one or more of BCL9, CD44, Axin2, cMyc,LGR5, VEGFA, Sox2, Oct4, Nanog, and/or active β-catenin. As used herein,active β-catenin refers to non-phosphorylated form of β-catenin.

Various known methods can be used to measure the gene expression leveland/or protein level of such biomarkers. For instance, a sample from asubject treated with the polypeptide or pharmaceutical composition canbe obtained, such as biopsy of a tumor, blood, plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes, orspleen. In some embodiments, the sample is a biopsy of a tumor in asubject. The sample obtained from a subject may be stained with one ormore antibodies or other detection agents that detect such biomarkers.The samples may also or alternatively be processed for detecting thepresent of nucleic acids, such as mRNAs, encoding the biomarkers viae.g., rt-qPCR methods.

In some embodiments, a reduced gene expression level and/or proteinlevel of BCL9, CD44, Axin2, cMyc, LGR5, VEGFA, Sox2, Oct4, Nanog, and/oractive β-catenin indicates treatment efficacy of a polypeptide orpharmaceutical composition described herein. The expression level ofsuch biomarker may be measured after e.g., 1 day, 2 days, 3 days, 4days, 5 days, one week, or two week of administration of the polypeptideor pharmaceutical composition, or any time period inbetween. In someembodiments, a method is disclosed comprising measuring the level of oneor more of the biomarkers after one or more rounds of administration ofa polypeptide or pharmaceutical composition described herein. In someembodiments, the method further comprises continuing to administer thepolypeptide or pharmaceutical composition if the biomarker levels arereduced. In some embodiments, the method further comprises administeringan increased dosage of a polypeptide or pharmaceutical compositiondescribed herein if the biomarker levels are not reduced, or increasingthe frequency of subsequent administrations. In some embodiments,treatment is discontinued if biomarker levels are not reduced after theinitial administration. In various embodiments, biomarker levels arealso measured before a first administration of the polypeptide orpharmaceutical composition described herein, and compared to levelsafter one or more rounds of administration, wherein treatment efficacyand continued treatment steps are determined based on the change inbiomarker level(s) from the level(s) prior to administration.

In some embodiments, an increased gene expression level and/or proteinlevel of BCL9, CD44, Axin2, cMyc, LGR5, VEGFA, Sox2, Oct4, Nanog, and/oractive β-catenin indicates that a subject would benefit from treatmentwith a polypeptide or pharmaceutical composition described herein, thana subject who does not have increased gene expression levels and/orprotein levels. In some embodiments, methods of treatment are disclosed,comprising selecting patients having increased biomarker levels andadministering a polypeptide or pharmaceutical composition describedherein.

In certain embodiments, a subject having elevated level of gene and/orprotein expression of BCL9, CD44, Axin2, cMyc, LGR5, VEGFA, Sox2, Oct4,Nanog, and/or active β-catenin is selected for treatment with apolypeptide or pharmaceutical composition described herein. In someembodiments, a subject suffering from a tumor is selected for treatmentafter obtaining a tumor sample from the subject and identifying anelevated gene and/or protein expression of BCL9, CD44, Axin2, cMyc,LGR5, VEGFA, Sox2, Oct4, Nanog, and/or active β-catenin. In someembodiments, a subject suffering from a tumor is selected for treatmentafter obtaining a tumor sample from the subject and identifying anelevated gene and/or protein level of BCL9. In some embodiments, asubject suffering from a tumor is selected for treatment after obtaininga tumor sample from the subject and identifying an elevated gene and/orprotein level of CD44. In some embodiments, a subject suffering from atumor is selected for treatment after obtaining a tumor sample from thesubject and identifying an elevated gene and/or protein level of activeβ-catenin.

5. Dosage

Dosage regimens may be adjusted to provide an optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage.

The term “dosage unit form” refers to physically discrete units suitedas unitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit formsprovided herein are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals. An exemplary, non-limiting range for atherapeutically or prophylactically effective amount of a bindingprotein provided herein is 0.1-20 mg/kg, for example, 1-10 mg/kg. It isto be noted that dosage values may vary with the type and severity ofthe condition to be alleviated. It is to be further understood that forany particular subject, specific dosage regimens may be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

6. Kit

Also disclosed herein are kits for performing methods described herein.In various embodiments, a kit for manufacturing a polypeptide describedherein is provided. In some embodiments, the kit comprises a polypeptidethat is capable of undergoing a reaction to from one or more hydrocarboncrosslinkers. In some embodiments, the kit comprises a metal catalystfor performing metal-mediated ring-closing olefin metathesis.

In various embodiments, a kit for treating a disease in a subject isalso provided. In some embodiments, the kit is for treating cancer in asubject. In some embodiments, the kit comprises a polypeptide orpharmaceutical composition disclosed herein. In some embodiments, thepolypeptide in the kit is capable of undergoing a reaction to from oneor more hydrocarbon crosslinkers. In some embodiments, the polypeptidein the kit has one or more hydrocarbon crosslinkers. In someembodiments, the kit further comprises at least one additional agentthat can be administered to a subject.

In various embodiments, a kit for detecting and/or treating a subjecthaving a tumor exhibiting elevated gene and/or protein expression ofBCL9, CD44, Axin2, cMyc, LGR5, VEGFA, Sox2, Oct4, Nanog, and/or activeβ-catenin in a subject is provided. In some embodiments, the kitcomprises agents for detecting the gene and/or protein expression ofBCL9, CD44, Axin2, cMyc, LGR5, VEGFA, Sox2, Oct4, Nanog, and/or activeβ-catenin. In some embodiments, the kit further comprises a polypeptideor pharmaceutical composition disclosed herein. In some embodiments, thepolypeptide in the kit is capable of undergoing a reaction to from oneor more hydrocarbon crosslinkers. In some embodiments, the polypeptidein the kit has one or more hydrocarbon crosslinkers. In someembodiments, the kit further comprises at least one additional agentthat can be administered to a subject.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs.

The articles “a” and “an” refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. For example, “anelement” means one element or more than one element.

The term “or” means, and is used interchangeably with, the term“and/or,” unless context clearly indicates otherwise. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”are not limiting. Any range described herein will be understood toinclude the endpoints and all values between the endpoints.

To the extent that the term “contain,” “include,” “have,” or grammaticalvariants of such term are used in either the disclosure or the claims,such term is inclusive in a manner similar to the term “comprising” as“comprising” is interpreted when employed as a transitional word in aclaim. The term “including” or its grammatical variants mean, and areused interchangeably with, the phrase “including but not limited to.”

The term “about” means a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight, or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length. When the term “about” is used in conjunctionwith a numerical range, it modifies that range by extending theboundaries above and below the numerical values set forth. In general,the term “about” is intended to modify a numerical value above and belowthe stated value by a variance of ≤10%.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinare obvious and may be made using suitable equivalents without departingfrom the scope of the embodiments disclosed herein. Having now describedcertain embodiments in detail, the same will be more clearly understoodby reference to the following examples, which are included for purposesof illustration only and are not intended to be limiting.

EXAMPLES Example 1. Generation of Stabilized BCL9 Peptides

Methods of synthesizing hydrocarbon crosslinkers using modified Alaresidues (α,α-disubstituted amino acids such as α-methyl, α-alkenylamino acids) are known in the art. See e.g., US20140113857 and Kim 2011.Each stabilized peptide used in the following examples was generated bya one on-resin synthesis method. Peptide elongation was performed onresin to generate each polypeptide, followed by a ring closingmetathesis.

Hydrocarbon crosslinkers with different lengths, such as an 8-carboncrosslinker and an 11-carbon crosslinker, can be generated using anα-methyl, α-alkenyl amino acid with an alkenyl chain of suitable length.For instance, (S)2-(4′pentenyl)Ala was incorporated into a polypeptideto construct a stabilized polypeptide having an 8-carbon crosslinkerwith an S-configuration on both ends. (R)2-(4′-pentenyl)Ala wasincorporated into a polypeptide to construct a stabilized polypeptidehaving an 8-carbon crosslinker with an R-configuration on both ends. Fora stabilized polypeptide having an 8-carbon crosslinker with anS-configuration on one end and an R-configuration on the other end,(S)2-(4′-pentenyl)Ala and (R)2-(4′-pentenyl)Ala were used, respectively.To construct a stabilized polypeptide having an 11-carbon crosslinkerwith an S-configuration on one end and an R-configuration on the otherend, (R)2-(7′-octenyl)Ala and (S)2-(4′-pentenyl)Ala were used,respectively.

Each polypeptide was purified using standard high-performance liquidchromatography (HPLC) protocols. A Zorbax C18 reverse-phase column,9.4×250 mm (Agilent, pore size 80 Å, particle size 3.5 μm) was used. Thesolvents used were A: water, 0.1% (vol/vol) TFA; B: acetonitrile, 0.1%(vol/vol) TFA. The flow rate was 4 ml/min. The gradient was 10-100%(vol/vol) B over 30 min; 100% B over 5 min; 100-10% (vol/vol) B over 4min; 10% (vol/vol) B over 1 min. The injection volume was 100-400 μl.The wavelength (nm) was 280 (for Fmoc-, Trp- or Tyr-containingpeptides), or 220 (for others).

Example 2. Mapping of Functional Domains of the HD2 Domain of BCL9

As the Wnt signaling pathway impacts signaling in cancer and otherdiseases, stabilized peptides containing the HD2 domain of the BCL9peptide were investigated. A BLAST search indicated that the HD2 domainof human BCL9 protein is unique and therefore peptides derived from thisdomain should be specific for inhibiting the effects of BCL9.

To further identify the core functional domain of the HD2 domain of BCL9protein that binds to β-catenin, multiple lead optimization studies wereperformed in a series of three steps: (i) domain mapping of thefull-length HD2 domain of human BCL9 protein; (ii) point mutationswithin the potential core functional domain; and (iii) terminalmodification and staple site optimization. Two biochemical assays fordetecting the binding between polypeptides and β-catenin were developed.The first assay was a Homogeneous Time Resolved Fluorescence (HTRF)binding assay (Cisco), in which a biotinylated polypeptide wasconjugated with streptavidin-XL655 fluorescence and the histidine tag ofβ-catenin protein was conjugated with Eu-labeled monoclonal antibody(mAb). The second assay was an Amplified Luminescence ProximityHomogeneous Assay (ALPHA) screening assay (Perkin Elmer), in which abiotinylated polypeptide was conjugated with streptavidin coated donorbeads and β-catenin protein was conjugated to protein A-coated acceptorbeads through an anti-O-catenin antibody. In this binding assay, a PEGlinker was used to conjugate the biotinylated peptide to the beads,allowing more room for BCL9 derived polypeptides to bind to β-catenin.As assessed in the ALPHA screen assay, the K_(D) of the full-length HD2domain of human BCL9 protein to β-catenin was 20 nM, with a 180-foldsignal versus background ratio, resulting in a significant improvementover other known methods of detecting binding affinity. See e.g., Zhanget al., Analytical Biochemistry 469: 43-53 (2015) and Kawamoto et al.,Biochemistry 48(40):9534-9541 (2009).

Domain mapping was done by constructing twenty overlapping stapledpolypeptides (7 or 8-mers) spanning the HD2 domain of human BCL9.Polypeptides having a length of 8-20 amino acids are typically used asinhibitors of protein interactions as they have an additional advantageof being low-cost. As shown in FIG. 2A, this process involved thegeneration of small stabilized polypeptides and the use of pointmutations of selected amino acids of the HD2 domain to verify thesmallest functional domain of the HD2 domain. As seven amino acids isthe shortest sequence to form a single i, i+4 staple, sequentialstabilized peptides with a length of seven or eight amino acids weregenerated from amino acids 351 to 374 of human BCL9 protein. Table 2below summarizes each polypeptide generated for domain mapping andindicates which amino acid residues were substituted with(S)2-(4′pentenyl)Ala to further generate an 8-carbon crosslinker. Xaa₁and Xaa₂ in Table 2 represent (S)2-(4′pentenyl)Ala. Each polypeptidelisted in Table 2 was constructed and crosslinked according toExample 1. All the polypeptides have an 8-carbon crosslinker(—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—) with an S-configuration on both ends.The N-terminus of each polypeptide is modified with an acetyl groupwhile the C-terminus of the polypeptide is modified with an NH₂ group. Bin Table 2 represents norleucine.

TABLE 2 Polypeptides generated for domain mapping SEQ Amino AcidCorresponding ID Sequence position NO: (WX No.) within BCL9  60 LXaa₁QEQ351-357 Xaa₂E (WX-002)  61 SXaa₁EQLXaa₂ 352-358 H (WX-003)  62QXaa₁QLEXaa₂ 353-359 R (WX-004)  63 EXaa₁LEHXaa₂ 354-360 E (WX-005)  64QXaa₁EHRXaa₂ 355-361 R (WX-006)  65 LXaa₁HREXaa₂ 356-362 S (WX-007)  66EXaa₁RERXaa₂ 357-363 L (WX-008)  67 HXaa₁ERSXaa₂ 358-364 Q (WX-009)  68RXaa₁RSLXaa₂ 359-365 T (WX-010)  69 EXaa₁SLQXaa₂ 360-366 L (WX-011)  70RXaa₁LQTXaa₂R 361-367 (WX-012)  71 SXaa₁QTLXaa₂D 362-368 (WX-013)  72LXaa₁TRLXaa₂I 363-369 (WX-014)  73 QXaa₁LRDXaa₂Q 364-370 (WX-015)  74TXaa₁RDIXaa₂R 365-371 (WX-016)  75 LXaa₁DIQXaa₂B 366-372 (WX-017)  76RXaa₁IQRXaa₂L 367-373 (WX-018)  77 DXaa₁QRBXaa₂F 368-374 (WX-019)  78LRXaa₁IQRXaa₂ 366-373 L (WX-020) 105 LSQEQLEHRERSL 351-374 Xaa₁TLRXaa₂IQRBLF (WX-001)

As shown in FIG. 2B, all the stabilized polypeptides listed in Table 2were simultaneously tested in a cell viability assay. Colo320DM cellswere selected for this assay as the proliferation of this particularcell line is dependent on BCL9 and β-catenin. ICG001, a knownWnt/β-catenin small molecule inhibitor, was used as a positive control.The cell viability at each treatment condition was analyzed usingCellTiterGlo luminescent cell viability assay (Promega) according to themanufacturer's protocols.

As shown in FIG. 2B, ICG001 was capable of inducing cell death in a dosedependent manner. WX-020, comprising the distal portion of the HD2domain, was also efficient in inhibiting cell growth, comparable toICG001. The IC₅₀ value of WX-020 in this assay was 12.4 μM. The effectof WX-020 (comprising LRDIQRBL (SEQ ID NO: 106)) was superior to WX-018(comprising RDIQRBL (SEQ ID NO: 107)), which does not have Leu at theN-terminus, suggesting that the core functional domain that binds toβ-catenin involves the stretch of 8 amino acid residues encompassed byWX-020. Also, this improved potency of WX-020 indicates that a shortstabilized polypeptide derived from the distal portion of the HD2 domainis sufficient to retain the ability to bind β-catenin and thereforeblock the interaction between BCL9 and β-catenin. The polypeptides werealso tested in a cell-based Wnt transcription inhibition assay usingGeneBLAzer® beta-lactamase (bla) reporter assay (invitrogen), asdescribed in Example 4.1. In this assay, HCT116 cells were selectedbecause the cells are known to have aberrant Wnt signaling activationdue to the presence of a β-catenin mutation. Consistent with the resultsfrom the cell viability assay, WX-020 was the most effective polypeptidein inhibiting the Wnt transcription (FIG. 2C).

Furthermore, as shown in FIG. 3 , when tested in the same cell viabilityassay, the effect of WX-020 exceeded that of full length HD2 domain(WX-001; “SAH-BCL9 #1”), indicating that not all polypeptides comprisingthe core functional domain are necessary equally effective in blockingthe interaction between BCL9 and β-catenin, dependent on the presence ofother sequence elements or chain modifications. WX-020 was also testedin the ALPHA screen as described above and shown to have a K_(D) valueof 80 nM. WX-020 was tested in a Wnt transcription assay following theprocedures described in Example 4.1 and shown to have an IC₅₀ value of1580 nM.

Point mutations altering amino acids of the distal portion of the HD2domain also reduced the efficacy of stabilized peptides, indicating thatthe distal region of the HD2 domain contains motifs of BCL9 involved inbinding to β-catenin. In particular, Structure Activity Relationships(SAR) evaluation of the WX polypeptides indicated that the hydrophobicamino acids on the WX polypeptides mediate the binding to β-catenin.

Example 3. Stabilized Polypeptides Comprising the Core Functional Domain

Four additional stapled polypeptides with varying lengths of amino acidswere generated, comprising the core functional domain identified inExample 2. The stapled polypeptides were further modified by addingknown peptide tags or terminal modifications to improve permeability andsolubility, such as Ant8, TAT, 03 Ala)-(β-Ala). Four additionalstabilized polypeptides corresponding to SEQ ID NOs: 108-111 weregenerated using the standard peptide synthesis protocol set forth inExample 1 (Table 3; see also Table 1). See also Kim 2011. For instance,to construct WX-024 as shown in FIG. 1A, a hydrocarbon crosslinker wasgenerated between the two Xaa amino acids of SEQ ID NO: 103 usingruthenium-mediated ring-closing olefin metathesis. Following the ringclosure metathesis, the polypeptide was deprotected and released. Theresulting stabilized polypeptide comprises a hydrocarbon crosslinker of—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂— with an S-configuration on both ends.WX-024 contains an acetyl-group at the N-terminus and 2-Naphthylalanineat the C-terminus. The carboxyl group of the 2-Naphthylalanine at theC-terminus is further modified with NH₂. WX-021, WX-022, and WX-023share the same sequence as WX-020, but modified with a differentchemical moiety at the C-terminus (2-Naphthylalanine and two units ofβ-alanines with NH₂ at the C-terminus, two units of β-alanines with NH₂at the C-terminus, and 2-Naphthylalanine with NH₂ at the C-terminus,respectively). All the polypeptides were further purified according tothe protocol described in Example 1.

Table 3 summarizes the four polypeptides comprising the core functionaldomain and indicates which amino acid residues were substituted with(S)2-(4′pentenyl)Ala to further generate an 8-carbon crosslinker (Xaa₁and Xaa₂=(S)2-(4′pentenyl)Ala).

TABLE 3Additional stabilized polypeptides derived from the HD domain of BCL9protein SEQ Corresponding ID Amino Acid Sequence position withinN-terminus C-terminus NO: (WX No.) BCL9 Modificiation Modification 108LRXaa₁IQRXaa₂L 366-373 Ac 2-Nal-ß- (WX-021) Ala-ß-Ala- NH₂ 109LRXaa₁IQRXaa₂L 366-373 Ac ß-Ala-ß- (WX-022) Ala-NH₂ 110 LRXaa₁IQRXaa₂L366-373 Ac 2-Nal-NH₂ (WX-023) 111 LQTLRXaa₁IQRXaa₂L 363-373 Ac 2-Nal-NH₂(WX-024)

The additional polypeptides were tested in a CellTiterGlo luminescentcell viability assay (Promega). ICG001 was used as a positive control inthis assay. As shown in FIG. 4A, while all the tested polypeptidesincluding WX-020 were capable of inhibiting cell growth in a dosedependent manner, WX-024 (and WX-021) was particularly effective in thisassay. In particular, the IC₅₀ value of WX-024 in this assay was largelyimproved as compared to WX-020, demonstrating that WX-024 comprising thecore functional domain is particularly effective in inhibiting cellviability and Wnt transcription as compared to other polypeptidescomprising the same core functional domain.

Some preliminary studies on solubility, stability and salt selectionwere performed with WX-024 in collaboration with ChemPartner. As shownin FIG. 4B, the acetate salt form of WX-024 was tested in a cell-basedWnt transcription inhibition assay and effectively inhibited Wnttranscription (IC₅₀=292 nM). Likewise, FIG. 4C shows that thehydrochloric salt also effectively inhibited Wnt transcription in thesame assay (IC₅₀=313 nM). WX-024 was soluble in water, DMSO, and PBS at1 mg/mL, and the trifluroacetic acid salt form of WX-024 was stable at2-8° C. and at room temperature for at least one month. In addition, thesalt form of WX-024 was active in various in vitro cell viabilityassays.

Example 4. In Vitro Profile of Stabilized Polypeptides

Binding of BCL9 to β-catenin is known to activate Wnt signaling. BecauseWnt/β-catenin activity regulates a large range of cell signals, avariety of assays can be used to measure activity of this pathway.WX-021 and WX-024 were assessed in a variety of cellular assays toassess its in vitro capability of modulating Wnt/β-catenin signaling.

Example 4.1. Cellular Activity of Stabilized Polypeptides

The cellular activity of WX-021 and WX-024 were measured via aGeneBLAzer® beta-lactamase (bla) reporter assay (invitrogen). ICG001 wasused as a comparison. The GeneBLAzer uses CellSensor™ LEF/TCF-blaHCT-116 cells that contain a beta-lactamase (BLA) reporter gene underthe control of the β-catenin/LEF/TCF response element stably integratedinto HCT-116 cells, as shown in FIG. 5A. These cells constitutivelyexpress beta-lactamase and can be used to detect agonists andantagonists of the Wnt/β-catenin signaling pathway. The GeneBLAzer assayprovides a ratiometric reporter response involving a two-color(blue/green) readout of stimulated and unstimulated cells.

LEF/TCF-bla HCT-116 cells were plated in assay medium (Invitrogen) onclear-bottom plates and incubated overnight at 37° C. Cells were thentreated with a vehicle control (0.05% DMSO in water) or a range of dosesof WX-021, WX-024, or ICG001 up to 10 μM for 5 hours. Cells were nextincubated with the Wnt agonist, mWnt3a (provided in the GeneBLAzer kit)for 5 hours at 37° C. Substrate mixture (containing LiveBLAzer™ B/Gsubstrate that employs CCF4-AM, a FRET substrate for beta-lactamase) wasadded to the wells, and the plate was incubated for 2.5 hours at roomtemperature in the dark. Black-walled, clear-bottom, 384-well plates(Corning Costar) were used with scanning done with a Spectramax M2.Plates were scanned first (Scan 1 in the blue channel) with excitationfilter 409/20 nm and emission filter 460/40 nm. Plates were then scanned(Scan 2 in the green channel) with excitation filer 409/20 nm andemission filter 530/30 nm. Fluorescence emission values at 460 nm and530 nm were obtained, and inhibition of the 460/530 ratios was used todetermine the percent inhibition (% inhibition) of Wnt/β-cateninsignaling by WX-024 using data analysis protocols from the manufacturer(Life Technologies).

As shown in FIGS. 5B-5D, increasing concentrations of WX-021 and WX-024produced greater inhibition of Wnt signaling in this reporter assay, ascompared to ICG001. The IC₅₀ of WX-021 and WX-024 calculated from thisassay were 764 nM and 191 nM, respectively. These stabilizedpolypeptides were more potent inhibitors of Wnt/β-catenin signaling thanthe known Wnt inhibitor, ICG001 (IC₅₀=1060 nM). Another Wnt inhibitor,LGK-974 (also known as Porcupine inhibitor), was tested in the sameassay. LGK-974 inhibits Porcupine, a membrane bound O-acyltransferasethat mediates palmitoylation of Wnt family proteins, which is requiredfor secretion and functional activation of Wnt protein. Liu et al., ProcNatl Acad Sci USA (2013) 110: 20224-20229. WX-024 also showed better invitro potency targeting Wnt/β-catenin transcription than LGK-974(IC₅₀>10 μM), which was expected, since LGK-974 targets extracellularWnt signaling and does not directly inhibit β-cat transcription (FIG.5E).

Overall, while WX-021 and WX-024 were both effective in suppressing Wntsignaling pathway, the effect of WX-024 showed improved functionalproperties in both cell viability assay (FIG. 4 ) and Wnt transcriptionassay (FIG. 5 ) as compared to WX-021. Thus, WX-024 was selected forfurther characterization in subsequent in vitro and in vivo studies.

Example 4.2. Binding Affinity of WX-024 to B-Catenin

The ability of WX-024 to bind β-catenin was evaluated in a homogenoustime resolved fluorescence (HTRF) assay (Cisbio). HTRF assays provide ameans of assaying protein-protein interactions in a high-throughputformat, as discussed in Degorce et al., Curr Chemical Genom. 3, 22-32(2009). In an HTRF assay to measure a protein-protein interaction,binding of 2 proteins brings an HTRF donor and acceptor fluorophore intoclose proximity and generates the fluorescence energy transfer (FRET)signal. Each protein of the interaction is associated with the donor orthe acceptor by an antibody reaction.

FIG. 6A provides a schematic to depict the basis of the HTRF assay. Inthe HTRF assay for β-catenin (Cisbio), β-catenin is bound by anXL665-coupled antibody (ab) against β-catenin. See Degorce (2009).β-catenin bound to an XL665-coupled antibody can interact with itsbinding partners, such as BCL9 or BCL9 peptides.

For the HTRF assay, the β-catenin binding partner, WX-024, was coupledto biotin. After incubation of β-catenin and WX-024, the reaction wasthen incubated with streptavidin (SA) bound to europium cryptate(SA-europium cryptate). SA is a high-affinity binding partner to biotinand will tightly bind the SA-europium cryptate to the biotinylatedstabilized polypeptides. Europium cryptate is an energy donor for anHTRF reaction. When not closely bound, there is no energy transferbetween europium cryptate and XL665. However, when europium cryptate andXL-66 are in close proximity, such as when there is successful bindingof biotinylated WX-024 to the β-catenin that is bound by the XL665antibody, there is an energy transfer that can be measured.

Histidine-tagged β-catenin, BCL9 peptide labeled with biotin, europium(Eu) labeled monoclonal antibody for histidine tag, and SA-XL665 wereused in the HTRF assay. Buffer for the assay contained 50 mM MES pH 6.5,1501111\1 NaCl; 0.1% BSA, 1 mM DTT, and 0.1% Tween 20. Five μL ofHis-tag protein was added to plate. Five μL of the biotin-labeledpeptide at various concentrations was added to the same well. Ten μL ofEu-labeled monoclonal antibody and SA-XL644 detection mixture wereadded. The reaction was incubated at room temperature for 1 hour andthen the plate was read.

The results obtained from WX-024 are shown in FIG. 6B. The K_(D) valueof WX-024 calculated from the results was 4.21 nM.

Example 4.3. Comparison of WX-024 and ICG001 in a Cell Viability Assay

FIG. 7 presents the data on viability of HCT116 cells treated withincreasing concentrations of either WX-024 or ICG001 using aCellTiterGlo luminescent cell viability assay (Promega) using standardmanufacturer's procedures. Both WX-024 and ICG001 were able to inhibitcell growth as measured by the viability assay. In particular, the IC₅₀value of WX-024 calculated from this assay was 1.92 μM, lower than thatof ICG001 (IC₅₀=2.17 μM).

Overall, the data indicate that WX-024 has a robust in vitro profileconsistent with inhibition of the Wnt signaling pathway.

Example 4.4. Comparison of WX-024 and Other Known ChemotherapeuticAgents

WX-024 was also tested in a Colo320DM cell viability assay and comparedto other chemotherapeutic agents. WX-024 was approximately 12-fold moreeffective in inhibiting cell growth of a BCL9/β-catenin dependent cellline than ICG001, LGK-974 (FIG. 8A) or Erlotinib (FIG. 8B).

WX-024 also offered benefits and improvements when used in combinationwith other known chemotherapeutic agents. In particular, as shown inFIG. 8C, WX-024 was tested in combination with a widely usedchemotherapy drug in a colorectal cancer, 5-fluorouracil (5-FU), andshowed that the combination therapy showed a significantly improvedability to inhibit cell growth (IC₅₀=1 μM) as compared to 5-FU treatmentalone (IC₅₀=12.1 μM).

Example 4.5. Specificity of WX-024

As discussed in Example 4.1, WX-024 inhibited Wnt transcription in acell-based assay. However, WX-024 did not produce any significantinhibition in other transcription assays, such as JAK/STAT (SIE-blaME-180 cell line), PI3K/AKT/FOXO (TREx FOXO3-DBE-bla HeLa cell line),TGF-beta (SBE-bla HEK 293T cell line), or TNF-alpha/JNK (AP1-bla ME-180cell line) pathways (Life Technologies), showing an IC₅₀ value greaterthan 10 μM in all these transcription assays. In these assays, thetrifluoroacetic salt form of WX-024 was used. The data, summarized inTable 4 below, demonstrate that WX-024 is an inhibitor that specificallytargets the Wnt/β-catenin pathway.

TABLE 4 Evaluation of WX-024 in additional pathways Pathway Cell LineTested Stimulation IC50 (nM) JAK/STAT SIE-bla ME-180 IL-6 >3160PI3K/AKT/FOXO3 TREx FOXO3- Insulin >1000 DBE-bla HeLa TGF-beta SBE-blaHEK 293T TGF-beta 1 >3160 TNP-alpha/JNK AP1-bla ME-180 TNF-alpha >1000Wnt/Beta-Catenin LEF-TCF-bla HCT116 None 191 nM

Example 5. In Vivo Profile of Stabilized Polypeptide

Based on the robust in vitro profile of WX-024, experiments wereperformed in mice to determine the plasma pharmacokinetic parameters. Inaddition, WX-024 was evaluated in a mouse xenograft model to test forefficacy in inhibiting tumor growth.

Example 5.1. Pharmacokinetic Profile of WX-024 in Mice

The pharmacokinetic (PK) profile of WX-024 was assessed followingintravenous (i.v.) and intraperitoneal (i.p.) injections in male ICRmice (an outbred strain). The tail vein was used for i.v. injections.The doses of WX-024 assessed with 1 mg/kg (i.v.) and 5 mg/kg (i.p. andi.v.). Following injections, blood samples were obtained via theretro-orbital vein at 5 min, 1, 2, 4, 6, 8, 12, and 24 hours postdose.Plasma was obtained from the blood samples following mixing with sodiumheparin and centrifugation.

The concentration of WX-024 (ng/ml) was determined using a LC/MS. LCanalysis was done using an Agilent 100 HPLC. MS analysis was done usingan AB Sciex API 4000. Calibration standards were prepared by serialdilution. The concentrations of WX-024 (ng/ml) over time are shown inFIG. 9A.

The maximum observed concentration (C_(max)), terminal half-life(T_(1/2)), total body clearance (CL), volume of distribution (V_(z)),area under the curve from the time of dosing to the last measurableconcentration (AUC_(0-t)), area under the curve from the time of dosingextrapolated to infinity (AUC_(0-inf)), and bioavailability werecalculated using noncompartmental analysis modules in the FDA-certifiedpharmacokinetic program WinNonlin Professional v5.2 (Pharsight). Thesevalues are summarized in FIG. 9B. Of note, 5 mg/kg administeredintravenously produced the greatest C_(max) for WX-024 with a value of47354 ng/ml. This is equivalent to greater than 30 μM, which is 15 timesgreater than the in vitro IC₅₀ calculated in Example 4, indicating thatphysiological relevant concentrations of WX-024 can be achieved in theplasma.

The T_(1/2) of WX-024 with all dosing regimens was between 2.4-2.5hours, which indicates that there is a substantial period wherein activeWX-024 is present in the plasma. The bioavailability of WX-024 followingi.p. dosing is 73.6%. This value is not provided for i.v. dosing as allof the compound enters the plasma so the bioavailability is 100%. Thevalues for C_(max), Cl, V_(z), AUC_(0-t), and AUC_(0-inf) are all alsoconsistent with a profile that indicates adequate exposure to WX-024following i.p. and i.v. dosing to potentially modulate Wnt signaling.

A similar pharmacokinetic study of WX-024 was performed with a differentmouse strain (female balb/c mice, n=2 per treatment) and additionaladministration routs, including intramuscular and subcutaneous routes.Briefly, a mixed solvent of 10% DMSO and 90% deionized H₂O was used todissolve WX-024. The peptide solution was prepared at the fixedconcentration of 0.5 mg/ml, and then suitable amounts were administeredaccording to the dose. The same mixed solvent was used as a vehicletreatment. Blood samples (300 IA) were collected periodically from theretro-orbital vein at 0.25, 1, 2, 4, 6, 7, 12, and 24-hours post-dose(and 36 and 48 hours post-dose for intramuscular, subcutaneous, andintraperitoneal routes), followed by plasma separation for pendingbioanalysis by LC-MS/MS. Standard PK parameters were calculated bynon-compartmental analysis modules in WinNonlin Professional (v5.2,Pharsight, FDA-certified). As shown in FIG. 10A, the plasmaconcentrations of WX-024 followed a similar trend to what was shown inFIG. 9A. The mean pharmacokinetic parameters calculated from thisexperiment are shown in FIG. 10B and FIG. 10C. Good bioavailability anda long half-life were observed for WX-024, indicating that daily dosingof WX-024 is feasible. Of note, the plasma concentrations of WX-024remained stable for a long period when administered via intraperitoneal,intramuscular, subcutaneous routs, demonstrating that WX-024 can achievephysiologically relevant concentrations in vivo when administered invarious routes.

Overall, these data indicate that WX-024 has a PK profile suitable forstudying the in vivo effect of inhibiting the interaction of BCL9 andI3-catenin.

Example 5.2. Efficacy of WX-024 in a Mouse Xenograft Model of Cancer

Based on the favorable PK profile of WX-024 in mice, as described above,the efficacy of WX-024 was investigated in a mouse xenograft coloncancer model. The Colo320DM colon cancer model in BALB/c nude mice wasemployed, in which Colo320DM cells are injected subcutaneously intoBALB/c nude mice. Tumors form from the Colo320DM cells as the nude micedo not mount a sufficient immune response to clear these tumors.

Colo320DM tumor cells (ATCC) were cultured in RPMI1640 medium (GIBCO,Cat #31800022) supplemented with 10% heat-inactivated fetal bovine serumat 37° C. Harvesting of Colo320DM cells was done when cells were growingin an exponential growth phase. Female BALB/c nude mice (ShanghaiLaboratory Animal Center) were used for the study at 6-8 weeks of age.Animals had free access to water and irradiation sterilized foodthroughout the study period. Each mouse (n=16) was inoculatedsubcutaneously in the right flank region with 5×10⁶ Colo320DM tumorcells in 0.1 mL of PBS. Tumors were allowed to develop for 14 days afterinoculation, at which time the mean tumor size was 156.78 mm³. Duringthe study, the care and use of animals was conducted in accordance withthe Association for Assessment and Accreditation of Laboratory AnimalCare (AAALAC). Following inoculation of tumor cells, animals werechecked daily for morbidity and mortality. The body weight of eachanimal was monitored throughout the study.

After the 14-day period for tumor development, the mice were dividedinto 4 treatment groups: 5, 10, and 15 mg/kg WX-024 and vehicle controlgroups. 4% ethanol+8% Tween 80+88% 10 mM PBS was used as a vehicle. Alltreatments were administered once daily. The WX-024 solution wasdissolved in pure DMSO and then diluted with sterile water. The dosingvolume was adjusted for body weight such that the dosing volume for eachanimal was 0.05 mL/10 g of body weight for i.v. dosing. The treatmentperiod was 14 days.

All treatments were begun as daily i.v. dosing. It was found during thestudy that i.v. administration of 15 mg/kg WX-024 caused body weightloss with an average body weight loss of approximately 11% as comparedto a vehicle treated group after 3 days of treatment. The injectionmethod was changed to i.p. injection at Day 7 for the 10 mg/kg and 15mg/kg groups. Dosing via i.p. injection (with the dosing volume adjustedto 0.1 ml/10 g) was maintained for the 10 and 15 mg/kg groups from Day 7of the treatment period onwards, and the body weight of these animalsstabilized. Mice treated with vehicle control or 5 mg/kg WX-024 receivedi.v. injections throughout the study without an apparent effect on bodyweight. Body weight changes in mice during the treatment period areshown in FIG. 11 .

Tumor volumes were measured in two dimensions using a caliper at Days 0,3, 5, 7, 9, 11, 14 of the treatment period (all dosing starting after 14days of tumor development). The volume of the tumor was measured usingthe equation: V=0.5 a×b², wherein a and b are the long and shortdiameters of the tumor, respectively. Tumor volumes are expressed inmm³. At the end of the study (i.e., following 22 days of treatment) thetumor mass weight was measured.

FIG. 12A shows tumor volumes in the different treatment groups overtime. At Day 14, the tumor volume (mean±standard error of mean values)was 2575±382.86 mm³ for vehicle control-treated mice. The tumor volumesfor WX-024-treated mice were 2039±373.29, 1370±114.39, and 1157.6±99.04mm³ for the 5, 10, and 15 mg/kg treatment groups, respectively. Thetumor volumes of mice treated with 10 mg/kg or 15 mg/kg WX-024 weresignificantly smaller at Day 14 of treatment compared withvehicle-treated animals (P<0.05, Kruskal-Wallis analysis). Compared tothe vehicle-treated group, tumor volumes were 53.2% smaller for the 15mg/kg WX-024 group and 44.9% smaller for the 10 mg/kg WX-024 group atDay 14 of the treatment period. Significantly smaller tumor volumes forthe 10 mg/kg and 15 mg/kg groups compared with the vehicle-control groupwere also seen on Day 11 of treatment. A preliminary dose-responsecorrelation of WX-024 with predicted tumor size resulted in an IC₅₀value of 12.69 mg/kg.

FIG. 12B shows tumor mass weight (measured in grams) at the end of thestudy following 22 days of treatment with vehicle or WX-024. Tumor massof mice treated with 10 or 15 mg/kg WX-024 (using the i.v./i.p. protocoldescribed above) was substantially less than tumor mass ofvehicle-treated mice. These data confirmed that treatment with either 10or 15 mg/kg inhibited tumor growth in the Colo320DM colon cancer modelin BALB/c nude mice.

At the conclusion of the Colo320DM model, tumor and intestinal sampleswere collected, fixed in formalin, and imbedded in paraffin usingstandard techniques. Four μm-thick section of tissue were prepared forimmunohistochemistry (IHC) staining. The tissue sections weredeparaffinized and rehydrated via sequential washing with xylene, gradedethanol and phosphate-buffered saline (PBS). Following deparaffinizationand rehydration, the tissue sections were subjected to hightemperature-induced epitope retrieval in target retrieval solution (10mM citrate buffer; pH 6.0).

Antibody binding was detected using an UltraVision Quanto DetectionSystem (Thermo, Cat #TL-060-QHD) kit following the manufacturer'sinstructions. The sections were treated with Hydrogen Peroxide Block andUltra V Block (provided in the kit) and then incubated with primaryantibody overnight at 4° C.: CD44 (Abcam, Cat #ab157107 diluted 1:500),Axin (LSBio, Rabbit IgG, Cat #LS-B7029, Lot #51907, Stock Conc.: 1000ug/ml), or VEGFA (VEGFA: LS Bio, Rabbit IgG, Cat #LS-B10263, Lot #65323,Stock Conc.: 1000 ug/ml). Rabbit IgG replaced the primary antibody inthe negative control. All sections were counterstained with hematoxylin,dehydrated and mounted. IHC images were taken with an Olympus CKX31SFreverse microscope.

FIG. 13A shows that the intestinal morphology was maintained in micetreated with vehicle, 10 mg/kg, or 15 mg/kg WX-024 for 22 days. FIG. 13Bshows staining of Axin 2, a downstream protein in the Wnt signalingpathway, indicating that 22 days of treatment with 15 mg/kg WX-024decreased Axin 2 staining as compared with the vehicle treatment.

FIG. 14A shows staining for CD44, another downstream protein in the Wntsignaling pathway, following 22 days of treatment either vehicle or 15mg/kg WX-024. Immunohistochemistry scores are denoted in the top-rightof each CD44 image. FIG. 14B shows the quantitative comparison of theaverage staining scores calculated from the CD44 staining images. Thesedata indicate that there was also a decrease in CD44 staining followingtreatment with WX-024.

Likewise, the tumor samples were stained for VEGFA and confirmed thatWX-024 was capable of suppressing VEGFA expression in tumors. FIG. 15Ashows representative images from two vehicle treated animals and twoWX-024 treated animals. The average immunohistochemistry scorescalculated from the staining images are summarized in 15B.

Thus, the data from the Colo320DM model indicate that WX-024 is capableof suppressing tumor growth and inhibiting Wnt/β-catenin signaling ascompared to a vehicle treatment. The effect of WX-024 on VEGFA alsoindicates that WX-024 may modulate the immune response via reducing VEGFexpression in tumors.

Example 5.3. Efficacy of WX-024 in a Mouse Syngeneic Model of Cancer(CT26)

The CT26 model of colon cancer was used as a model for investigatingtumor growth in mice with an intact immune system.

The effect of WX-024 on CT26 cell growth was first investigated invitro. CT26 cells were grown in F-bottom plates over 5 days. As shown inFIG. 16A, from day 1 (D1) to day 5 (D5), there was substantial growth ofCT26 cells, as measured by a luminescence cell viability assay.CellTiterGlo (Promega) cell viability assay. The ability of WX-024 (FIG.16B) and doxorubicin (FIG. 16C) to inhibit CT26 cell growth wasmeasured. Doxorubicin is a known inhibitor of cell growth and wasincluded as a control. FIG. 16D summarizes the in vitro data in CT26cells, showing that WX-024 and doxorubicin were both able to inhibitgrowth of CT26 cells, with WX-024 showing more potent activity. The IC₅₀value of WX-024 was 1.753 μM, much lower than that of doxorubicin, 7.520μM. The EGFR receptor antagonist, erlotinib, had an IC₅₀ greater than 2μM.

The syngeneic mouse model of colon cancer using CT26 was then assessed.Male Balb/c mice were injected subcutaneously in the right flank with5×104 CT26 cells each at approximately 5 weeks of age. Mice were thendosed with either vehicle or 20 mg/kg WX-024 i.p. daily. Eight mice wereincluded in each treatment group. Tumor growth was then assessed over 14days of dosing as shown in FIG. 17A. Tumor volume over time wassubstantially lower in mice treated with WX-024 compared to vehicle(P<0.001). FIG. 17B summarizes the average tumor growth inhibition rateof WX-024 throughout the experiment. The TGI after 3 days of dosing was89.2% while the TGI after 12 days of dosing was 70.0%. FIG. 18A showstumor growth rate of individual animals over 14 days of dosing withvehicle. FIG. 18B shows tumor growth rate of individual animals over 14days of dosing with 20 mg/kg WX-024. These data indicate that theinhibition of tumor growth seen in the CT26 model was consistent betweenanimals.

CD4⁺ T cell counts in the blood after 2 weeks of daily dosing witheither vehicle or 20 mg/kg WX-024 were also assessed. The whole bloodwas taken from each mouse. Lymphocytes were purified from the bloodsamples by Ficoll, and the cells were stained with fluorescent anti-CD4antibody. Data were measured by flow cytometry. FIG. 19A shows CD4⁺ Tcell counts presented as the percentage of total cells, indicating anincrease with dosing of WX-024. FIG. 19B shows relative T cell count pertotal cells in the blood sample of one specimen, indicating an increasewith dosing of WX-024. These data indicate an increase of T helper cellsand activation of the immune system following the treatment with WX-024.These data also indicate that combination therapy of WX-024 withimmunotherapy may be appropriate in cancers that do not respond toimmunotherapy alone, such as treatment with antibodies directed againstPD-1 or CTLA-4.

CD8⁺ T cell counts in the blood after 2 weeks of daily dosing witheither vehicle or 20 mg/kg WX-024 were also assessed. As describedabove, the whole blood was taken from each mouse. Lymphocytes werepurified from blood by Ficoll, and the cells were stained withfluorescent anti-CD8 antibody. Data were measured by flow cytometry.FIG. 20A shows CD8⁺ T cell counts presented as the percentage of totalcells, indicating an increase with dosing of WX-024. FIG. 20B showsrelative T cell count per total cells in blood samples, indicating anincrease with dosing of WX-024. These data indicate an increase incytotoxic T cells and activation of the immune system followingtreatment with WX-024, and further support the use of WX-024 togetherwith immunotherapy.

To further assess whether WX-024 is capable of modulating a tumormicroenvironment, tumor and blood samples were collected inanticoagulation tubes at the end of the study. Single cells separationwas performed and then cells were stained for FACS analysis. For cellsurface staining, CD45-PerCP-Cy5, CD4-FITC, CD8-PE-Cy7, and CD25-PE wereused. For intranuclear staining, cells were permeabilized overnight andthen stained with Foxp3-APC for 60 minutes. As shown in FIG. 21A,regulatory T cells in CD4⁺ T cell populations were significantly reducedin the tumor samples, while the ratio between CD8⁺ cytotoxic T cells andregulatory T cells was significantly increased (FIG. 21B). Arepresentative FACS analysis for a tumor sample in this experiment isshown in FIG. 21C and FIG. 21D.

FIG. 22A shows staining for active β-catenin, following 14 days oftreatment with either vehicle or 20 mg/kg WX-024. The averageimmunohistochemistry scores were calculated based on the stainingimages. FIG. 22B shows the quantitative comparison of the averagestaining scores between the vehicle treated group and the WX-024 treatedgroup. FIG. 22C shows staining for CD44, following 14 days of treatmentwith either vehicle or 20 mg/kg WX-024. These data indicate that WX-024was capable of suppressing active β-catenin expression in tumors.

The tumor samples were also stained for PD-L1 and the representativeimages from four vehicle treated mice and four WX-024 treated mice areshown in FIG. 23A. The quantitative comparison of averageimmunohistochemistry scores for each treatment group are shown in FIG.23B.

Overall, these data indicate that WX-024 stimulates T cell infiltrationto tumors and stimulates an immune reaction against the tumors. Thesedata also indicate that WX-024 modulates a tumor microenvironment tofavor such an immune reaction. Thus, a combination therapy of WX-024 andcheckpoint-blocking agents may produce a synergistic effect instimulating an immune reaction against a tumor through reduction ofregulatory T cells or dendritic cells present in the tumor. Furthermore,since WX-024 was capable of increasing CD8⁺ T cells in blood, it furtherindicates that WX-024 likely increases T cell infiltration in tumor andimmunogenicity against tumor.

Example 5.4. Efficacy of WX-024 in a Mouse Syngeneic Model of Cancer(B16F10)

The ability of WX-024 to suppress other types of tumors was alsoassessed. C57BL/6 mice were inoculated with B16F10 cells. Female C57BL/6mice aged 4-5 weeks were inoculated with B16F10 cells (2×10⁵ cells in0.05 ml per mouse) in the right flank. When the average tumor size ofthe mice reached 100 mm³, the mice were divided into two groups (N=6).The first group was administered 25 mg/kg WX-024 while the second groupwas treated with a vehicle. The treatment was intraperitoneallyadministered daily for 14 consecutive days. As shown in FIG. 24 , WX-024effectively reduced tumor growth as compared to vehicle treatment, againconfirming that WX-024 has a robust efficacy in suppressing tumor growthin vivo.

To further assess whether WX-024 is capable of inducing T cellinfiltration into tumors and modulating the tumor microenvironment,C57BL/6 mice were inoculated with B16F10 cells. When the average tumorsize of the mice reached 100 mm³, the mice were divided into two groups(N=3) and treated with a vehicle or 25 mg/kg WX-024. The treatment wasintraperitoneally administered daily for 12 consecutive days. At the endof the treatment, the mice were sacrificed and tumor and blood sampleswere collected in anticoagulation tubes. Single cell separation wasperformed for each sample and then stained for FACS analysis. For cellsurface staining, CD45-PerCP-Cy5, CD4-FITC, CD8-PE-Cy7, and CD25-PE wereused. The tumor samples were then stained with either an anti-CD4antibody or an anti-CD8 antibody to assess the presence of T cells inthe tumor samples. The tumor samples were also stained with ananti-CD194 antibody and anti-CD196 antibody to assess the presence of Thelper 17 cells in the tumor samples.

As shown in FIG. 25A, WX-024 increased the CD4⁺ positive cells presentin the tumor samples, as compared to vehicle, indicating that WX-024induces T helper cell infiltration into tumors. Likewise, FIG. 25Bdemonstrates that WX-024 increased the CD8⁺ positive cells present inthe tumor samples, indicating that WX-024 induces cytotoxic T cellinfiltration into tumors. As summarized in FIG. 25C, these resultsdemonstrate that overall, WX-024 is capable of inducing T cellinfiltration into tumors and thereby further elicits beneficial immunereactions specifically targeting the tumor mass. Overall, the data showsa tendency of WX-024 to increase CD8+, and CD4+ lymphocyte infiltrationin tumors, indicating that WX-024 induces a tumor microenvironment tofavor an immune reaction by altering compositions of T cells in and/oraround the tumor.

The effects of WX-024 on a tumor microenvironment were compared withthose of WX-039, a variant of WX-024. The process of constructing WX-039is described in Example 10. C57BL/6 mice were inoculated with B16F10cells. When the average tumor size of the mice reached 100 mm³, the micewere divided into three groups (N=3) and treated with a vehicle, 20mg/kg WX-024, or 60 mg/kg WX-039. The treatment was intraperitoneallyadministered daily for 12 consecutive days. At the end of the treatment,the mice were sacrificed and tumor and blood samples were collected inanticoagulation tubes. Tumor cells collected from the experiment werestained as described above and also with anti-CD205 antibody to assesswhether WX compounds are capable of modulating dendritic cells. As shownin FIGS. 26A and 26B, WX-024 significantly reduced CD205⁺ cells,confirming that WX-024 is capable of suppressing dendritic cells withina tumor. While WX-039 was also capable of suppressing dendritic cellspresent in tumor, the effect of WX-024 was more pronounced than WX-039.FIG. 26C also shows that while WX-039 increased T helper 17 cells intumor, WX-024 appeared to be more effective than WX-039. Consistent withCT26 animal model studies, these data indicate that WX-024 is capable ofstimulating an immune reaction against tumors and modulating the tumormicroenvironment to favor such an immune reaction. A representative FACSanalysis for a tumor sample in this experiment is shown in FIG. 26D.

In combination with other immunotherapy agents, such as an anti-PD-L1antibody or other antibodies that are capable of modulating T cellactivation, WX-024 could modulate regulatory T cells and increase theratio between CD8⁺ cytotoxic T cells and regulatory T cells. WX-024 incombination with those immunotherapy agents could also furthersynergistically decrease myeloid dendritic cells in tumor and therebyinduce a tumor microenvironment favoring an immune reaction against atumor.

Example 5.5. Efficacy of WX-024 in a Mouse Orthotopic Model of Cancer

Orthotopic mouse models are useful to visualize the effect of ananti-tumor compound on a tumor implanted in a mouse and to assess theeffect of the compound on tumor metastasis. An orthotropic modelutilizing the NCI-H1299-Luc cell line (non-small-cell lung carcinomacell line expressing luciferase) was selected for this experiment. Uponuptake of luciferin substrate, these cells are capable of producingbioluminescence. Proliferation of the NCI-H1299 cell line is dependenton Pygopus (Pygo) 2/β-catenin transcription activity, in which TCF-1transcription factors (BCL9, β-cat and Pygo) form a transcriptioncomplex to initiate transcription.

To assess the effect of WX-024 in an orthotopic animal tumor model,female balb/c nude mice were injected with NCI-H1299-Luc cells. The micewere about 5 weeks of age. NCI-H1299-Luc cells (5×10⁶) in 50 μL in PBSwere injected into each mouse lung with a syringe. After the cells wereadministered to the mice, the mice were divided into two groups (n=3each) and a vehicle or 15 mg/kg WX-024 was administered intravenouslydaily for 10 consecutive days.

The bioluminescence intensity (total Flux [p/s]) of each mouse wasmeasured periodically throughout the study according to manufacturerprotocol (Xenogen ivis imaging system, PerkinElmer). The average totalflux depicted in FIG. 27A indicates the average bioluminescence photonflux signal generated by tumor cells in the mice treated with thevehicle or WX-024. As shown in FIG. 27A, WX-024 almost completelyblocked tumor formation and growth in vivo, demonstrating the in vivoefficacy of WX-024. FIG. 27B shows the total flux of each mousemonitored in this experiment. The data indicate that the inhibitoryeffect of WX-024 on tumor formation and growth was consistent among themice. The data also indicate that WX-024 is capable of suppressing tumormetastasis. FIG. 27C shows a bioluminescence image of each mouse, takenthroughout the study. Mice #1-3 are from the vehicle treated group whilemice #4-6 are from the WX-024 treatment group.

Example 5.5. Efficacy of WX-024 in a Patient Derived Xenograft (PDX)Animal Model

To assess whether WX-024 is also applicable to tumor derived from humanpatients, a xenograft animal model inoculated with patient derived coloncancer cells was exposed to WX-024. Samples derived from four differentcolon cancer patients were screened for biomarkers that indicate anelevated level of canonical Wnt/β-catenin signaling. As shown in FIG.28A, the samples (#5-#8) were stained for β-catenin, BCL9, c-Myc, andCD44 expression and also stained for IgG as a control. The samples werealso stained for nuclear co-localization of BCL9 and β-catenin. Thesample derived from patient #8 showed elevated levels of all biomarkerstested in this experiment and therefore was selected for thepatient-derived xenograft animal study.

Male NOD/SCID mice at about 5 weeks of age were intravenously injectedwith colon cancer cells derived from patient #8. Briefly, the selectedCRC tumor samples from FIG. 28A were sliced into 3 mm³ fragments andsubcutaneously implanted onto the right flank of NOD/SCID mice. Whenmean tumor size reached about 135 mm³ (about 18 days aftertransplantation), the mice were divided into two groups (n=8 each) andadministered either a vehicle or 15 mg/kg WX-024. Each moue was given adaily intravenous administration for more than 31 days.

As shown in FIG. 28B, WX-024 effectively inhibited the tumor growth ascompared to vehicle. The average tumor growth inhibition rate of WX-024was 75.6% after 31 days of dosing. At the end of the study, the micewere sacrificed and tumor samples were collected. Tissue samples werefixed with 10% formalin and transferred into 70% ethanol, and thenprocessed following deparaffinization, antigen retrieval (190° C., 5min), endogenous peroxidase quenching (3% H2O2, RT, 5 min), blockingwith 5% FBS for 15 minutes at room temperature, primary antibodyincubation (room temperature, 1 h), secondary antibody incubation (roomtemperature, 30 min), color development with DAB (room temperature, 5min) and dehydration and mounting. Each tumor sample was stained forBCL9, β-catenin, and CD44 expression and the results are shown in FIGS.29A, and 29B. Of note, WX-024 was capable of reducing CD44 expression inthose tumor samples, confirming that WX-024 is capable of inhibitingWnt/β-catenin signaling.

These data indicate that WX-024 is an effective therapy for humanpatients. The data also suggest that various biomarkers such asβ-catenin, BCL9, c-Myc, and CD44 can be useful to determine whether agiven patient is particularly suitable for WX-024 treatment.

Example 5.6. Toxicology Study of WX-024

To assess the toxicity effect of WX-024, female balb/c mice of about 5weeks of age were treated with WX-024 for 14 consecutive days. The micewere divided into four groups (n=6 each) treated intravenously with adaily dose of vehicle, or 10 mg/kg, 15 mg/kg, or 20 mg/kg WX-024. At theend of the study, the mice were sacrificed and various major organs werecollected. The tissue samples were treated and processed as described inExample 5.5. The embedded tissue samples were sectioned and stained withH&E. Representative H&E staining images from the vehicle treated groupand 10 mg/kg WX-024 treated group are shown in FIG. 30A. The body weightof each moue was monitored throughout the study and the average bodyweight of each treatment group is shown in FIG. 30B. While there was aslight decrease at the beginning of the study, the average body weightof all three WX-024 treated groups stabilized and was maintainedthroughout the study. Likewise, while the food consumption of all WX-024treated groups was slightly decreased at the beginning of the study, itrecovered to near baseline levels with continued treatment. At the endof the study, blood from each mouse was also collected for serumchemistry and hematological analysis (complete blood cell countanalysis). FIG. 30C shows the blood cell count profiles of the vehicletreatment group and 20 mg/kg WX-024 treatment group. The profiles ofthese two groups did not differ significantly, indicating that WX-024induced no significant toxicity in this experimental condition.

The toxicokinetics of WX-024 were analyzed in female balb/c mice ofabout 5 weeks of age. The mice were treated intraperitoneally with 10mg/kg, 15 mg/kg, or 20 mg/kg WX-024 for 14 consecutive days. The micewere administered with WX-024 daily. FIG. 31A shows the plasmaconcentration change over 24 hrs after the first dosing of theexperiment at day 1 (n=2 each group). The mean toxicokinetic parameterscalculated at day 1 are summarized in Table 5 below.

TABLE 5 Mean toxicokinetic parameters of WX-024 after the first dosingTK parameters Unit IP-10 mg/kg IP-15 mg/kg IP-20 mg/kg T_(max) hr 1.502.17 2.67 C_(max) ng/mL 11390 9767 12733 Terminal t_(1/2) hr 4.52 7.596.47 AUC_(last) hr*ng/mL 114333 118033 162333 AUC_(INF) hr*ng/mL 117000129100 175333

FIG. 31B shows the plasma concentration change over 24 hrs after thelast dosing of the experiment at day 14. Table 6 summarizes the meantoxicokinetic parameters calculated at day 14. A toxicokinetic studyusing IP dosing (15 and 20 mg/kg) displayed comparable AUC valuesbetween Day 1 (118,033 and 162,333 hr*ng/mL, respectively) and Day 14(107,420 and 159,976 hr*ng/m, respectively), indicating thatdrug-induced immunogenicity caused by WX-024 is unlikely.

TABLE 6 Mean toxicokinetic parameters of WX-024 after the last dosing TKparameters Unit IP-10 mg/kg IP-15 mg/kg IP-20 mg/kg T_(max) hr 2.00 3.334.00 C_(max) ng/mL 11360 8748 13725 Terminal t_(1/2) hr 6.97 4.25 4.55AUC_(last) hr*ng/mL 37836 107420 159976 AUC_(INF) hr*ng/mL 41091 108735160881

Example 6. WX-024 Preclinical Study Summary

Overall, WX-024 showed an improved in vitro efficacy as compared otherknown Wnt inhibitors. For instance, when tested in a cell-based Wnttranscription assay using HCT166 cells, WX-024 exhibited an IC₅₀ valueof about 200 nM. WX-024 also specifically inhibited Wnt transcription,with the stapled polypeptide showing IC₅₀ values greater than 100 NM inother transcription assays such as JAK/STAT, TGF-0, PI3K/AKT/FOXO3, andTNF-α/JNK reporter assays.

The overall pharmacokinetic profile of WX-024 was also acceptable forproducing a physiologically relevant plasma concentration in vivo. Forinstance, when tested at 10 mg/kg by a subcutaneous administrationroute, the bioavailability was about 70%. In this condition, T_(max) was8 hours and T_(1/2) was 13 hours while C_(max) was 7.7 μM.

The in vivo efficacy of WX-024 was confirmed in multiple animal models.For instance, the average tumor growth inhibition rate of WX-024 wasabout 70-80% in those animal models when administered for 3-6 weeks.When animals were chronically dosed with WX-024, no particulardrug-related toxicity was observed.

Example 7. Additional Stabilized Polypeptide Comprising the CoreFunctional Domain

WX-035, comprising the core HD2 functional domain, was constructedaccording to the protocols described in Example 1. Briefly, a firsthydrocarbon crosslinker was generated between Xaa₁ and Xaa₂ amino acidsof SEQ ID NO: 104 and a second hydrocarbon crosslinker was generatedbetween Xaa₃ and Xaa₄ amino acids of SEQ ID NO: 104, usingruthenium-mediated ring-closing olefin metathesis as outlined in Kim2011. Following the ring closure metathesis, the polypeptide wasdeprotected and released. The resulting stabilized polypeptide comprisesa first hydrocarbon crosslinker of —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂— withan S-configuration on one end and an R-configuration on the other endand a second hydrocarbon crosslinker of —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—with an S-configuration on both ends. The chemical structure of WX-035is shown in FIG. 1B. The polypeptide was further purified according tothe protocol described in Example 1.

Example 8. In Vitro Profile of Stabilized Polypeptide

Following the procedure described in Example 2, the ability of WX-035 toinhibit cell growth was tested in a cell viability assay using Colo320DMcells. As shown in FIG. 32 , WX-035 exhibited an improved IC₅₀ value ascompared to WX-024.

Example 9. In Vivo Profile of Stabilized Polypeptide Example 9.1.Pharmacokinetic Profile of WX-035 in Mice

The pharmacokinetic profile of WX-035 was assessed following theprocedures described in Example 5.1. Briefly, the mice were administered1 mg/kg or 5 mg/kg intravenously, or 5 mg/kg intraperitoneally. Bloodsamples were collected at 15 min, 1, 2, 3, 6, 8, 12, and 24 hours postadministration. FIG. 33A shows the plasma concentration of WX-035 inmale ICR mice. The maximum observed concentration (C_(max)), terminalhalf-life (T_(1/2)), total body clearance (CL), volume of distribution(V_(z)), area under the curve from the time of dosing to the lastmeasurable concentration (AUC_(0-t)), area under the curve from the timeof dosing extrapolated to infinity (AUC_(0-inf)), and bioavailabilitywere determined and are summarized in FIG. 33B. Of note, the half-lifemeasurements for WX-035 indicate improved stability in the blood withT_(1/2) values of greater than 4 hours.

As done with WX-024, the pharmacokinetic profile of WX-035 was againanalyzed in female balb/c mice (n=3 per treatment). Two differentadministration routes, intravenous or intraperitoneal, were comparedusing 30 mg/kg WX-035. As shown in FIG. 34A, the pharmacokinetic profileof WX-035 was similar to that observed with male ICR mice. The meanpharmacokinetic parameters were calculated following the procedures inExample 5.1 and are summarized in FIG. 34B. As observed with male ICRmice, the half-life of WX-035 in this experiment was more than 9 hours.

These data are consistent with the hypothesis that stabilization of BCL9peptides with multiple crosslinkers may improve PK parameters.

Example 9.2. Efficacy of WX-035 in a Mouse Syngeneic Model of Cancer

The in vivo efficacy of WX-035 in treating a cancer was assessed in asyngeneic mouse model, following the procedures described in Example5.3. Trametinib, a known MEK inhibitor, was used as a comparison.Briefly, balb/c mice were inoculated with CT26 and treatment startedwhen tumor volume reached to about 100 mm³. The mice were then dividedinto three groups and began administered with a vehicle, 40 mg/kgWX-035, or 1 mg/kg trametinib for 7 consecutive days.

As shown in FIGS. 35A and 35B, WX-035 was capable of robustlysuppressing tumor growth as compared to trametinib and vehicletreatments. The average tumor growth inhibition rate of WX-035 was88.44% at the end of the study.

Example 9.3. Effect of WX-035 in T Cell Activation

At the end of the study described in Example 9.2, a tumor sample fromeach mouse was collected to assess the effect of WX-035 in modulating Tcell activation. The FACS analysis of each tumor sample was performedfollowing the procedure described in Example 5. A shown in FIG. 36A,treatment with WX-035 reduced the total amount of CD45⁺ cells. Becausethe total amount of haematolymphoid cells was reduced, the total amountof CD4⁺ or CD8⁺ T cells was also reduced (FIG. 36B). Likewise, Foxp3⁺CD25⁺ T cells were reduced by WX-035 treatment (FIG. 36C). However, asshown in FIG. 36D, WX-035 increased the ratio between cytotoxic T cells(CD8⁺ T cells) and Foxp3⁺ T cells (regulatory T cells) as compared tothe vehicle treatment. Therefore, these data suggest that WX-035 iscapable of reducing regulatory T cells in a syngeneic animal model.Furthermore, by increasing the ratio of cytotoxic T cells overregulatory T cells, WX-035 may induce a microenvironment that favors animmune reaction beneficial for tumor treatment. Of note, the samplescollected from this experiment were also assessed for LGR5 expression.As shown in FIG. 36E, WX-035 reduced the expression of LGR5 in intestinesamples collected from this experiment, confirming that WX-035 iscapable of inhibiting Wnt/β-catenin signaling in vivo.

Example 9.4. Toxicity Effect of WX-035

To assess the toxicity effect of WX-035, balb/c nude mice wereadministered with a vehicle, or 30 mg/kg, or 40 mg/kg WX-035, followingthe procedure described in Example 5.6. At the end of the study, themice were sacrificed and major organs were harvested for H&E staining.As shown in FIG. 37 , treatment with WX-035 did not cause anysignificant abnormality to major organs, indicating that WX-035 did notproduce a significant toxic effect.

Example 10. Additional Stabilized Polypeptides

Additional polypeptides derived from the HD2 domain of human BCL9protein were also constructed and the sequences of those polypeptidesare shown in Table 7 below. The polypeptides were constructed andpurified according to the protocol described in Example 1. WX-029comprises a 8-carbon hydrocarbon crosslinker between amino acids Xaa₁and Xaa₂. WX-037, WX-038, and WX-039 each comprises two hydrocarboncrosslinkers. In each polypeptide, a first 8-carbon crosslinker wasgenerated between Xaa₁ and Xaa₂ amino acids of its respective sequenceand a second 8-carbon crosslinker was generated between Xaa₃ and Xaa₄amino acids of its respective sequence. WX-037 is a polypeptide derivedfrom the HD2 domain of human BCL9 protein, wherein hydrophobic leucine(L) residues have been mutated to charged aspartic acid (D). WX-040comprises a 11-carbon crosslinker between amino acids Xaa₁ and Xaa₂.

TABLE 7Additional stabilized polypeptides derived from the HD domain of BCL9protein SEQ Corresponding ID Amino Acid Sequence position withinN-terminus C-terminus NO: (WX No.) BCL9 Modificiation Modification  97LQTLRXaa₁IQRXaa₂L 363-373 Ac 2-Nal-ß- (WX-029) Ala-ß-Ala- GRKKRRQRRRP Q 98 Xaa₁LQXaa₂LRXaa₃IQRX 362-373 Ac 2-Nal-ß- aa₄L (WX-036) Ala-ß-Ala-GRKKRRQRRRP Q  99 Xaa₁DQXaa₂DRXaa₃DQRX 362-374 Ac ß-Ala-ß- aa₄DH Ala-NH₂(WX-037) 100 Xaa₁LEXaa₂LRXaa₃IERX 362-373 Ac 2-Nal-ß- aa₄L Ala-ß-Ala-(WX-038) NH₂ 101 RXaa₁LQXaa₂LRXaa₃IQR 361-373 Ac 2-Nal-ß- Xaa₄LAla-ß-Ala- (WX-039) NH₂ 102 LQXaa₁LRDIQRXaa₂L 363-373 Ac 2-Nal-ß-(WX-040) Ala-ß-Ala- NH₂

Example 11. In Vitro Profile of Stabilized Polypeptides

One of the stabilized polypeptides generated in Example 10 was tested ina cell viability assay and compared against WX-035. WX-037 was selectedfor this comparison study. The cell viability assay was performed withCT26.WT cells (ATCC), following the procedures described in Example 2.Briefly, 4000 cells per well of each plate were seeded with 50 μLopti-MEM medium. Each polypeptide, WX-035 or WX-037, was diluted with 2%FBS containing opti-MEM medium using 1 μL of 200× concentratedpolypeptide in 99 μL of the medium. Each stock solution was diluted fortesting a wide range of concentrations. 50 μL of 2× concentratedpolypeptide was added to each well of the plate. Three days after thepolypeptide addition, the viability of cells was measured usingCellTiterGlo assay, according to the manufacturer's protocol.

As shown in FIGS. 38A and 38B, WX-035 more effectively inhibited cellviability in this condition (Ab IC₅₀=1.858 μM) than WX-037 (Ab IC₅₀>32μM). The data indicate that hydrophobic leucine residues furtherincrease the efficacy of a polypeptide derived from the HD2 domain ofhuman BCL9 protein. FIG. 38C summarizes the in vitro profiles of eachpolypeptide.

Example 12. In Vivo Profile of Stabilized Polypeptides Example 12.1.Pharmacokinetic Profiles of Stabilized Polypeptides in Mice

The pharmacokinetic profiles of the four additional polypeptidesconstructed in Example 10 were assessed following the proceduresdescribed in Example 5.1. WX-029 and WX-036 were administered 1 mg/kgintravenously to female balb/c nude mice. Blood samples were collectedat 15 min, 1, 2, and 4 hours post administration. FIG. 39A and FIG. 39Bsummarize the plasma concentration and the pharmacokinetic profilescalculated for WX-029. FIG. 40A and FIG. 40B summarize the plasmaconcentration and the pharmacokinetic profiles calculated for WX-036.

Similarly, WX-039 and WX-040 were also tested in female balb/c nude miceand assessed for their pharmacokinetic profiles. WX-039 was administeredat 5 mg/kg intravenously, 5 mg/kg intraperitoneally, or 10 mg/kgsubcutaneously. WX-040 was administered at 30 mg/kg intravenously or 30mg/kg intraperitoneally. Blood samples were collected at 15 min, 1, 2,4, 8, 24, 36, 48, and 72 hours post administration. FIG. 41A and FIG.41B summarize the plasma concentration and the pharmacokinetic profilescalculated for WX-039. FIG. 42A and FIG. 42B summarize the plasmaconcentration and the pharmacokinetic profiles calculated for WX-040. Ofnote, the half-life of WX-039 calculated in this experiment was about 52hours, when administered subcutaneously.

Example 12.2. Efficacy of WX-039 in a Mouse Syngeneic Model of Cancer(B16F10)

The ability of WX-039 to suppress tumor growth was assessed in theanimal model described in Example 5.4. Briefly, C57BL/6 mice wereinoculated with B16 cells. Female C57BL/6 mice aged 5 weeks wereinoculated with B16F10 cells (2×10⁵ cells in 0.05 ml per mouse) in theright flank. When the average tumor size of the mice reached 100 mm³,the mice were divided into two groups (N=3). The first group wasadministered 60 mg/kg WX-039 while the second group was treated with avehicle. The treatment was intraperitoneally administered daily for 12consecutive days. As shown in FIG. 43A, WX-039 reduced tumor growth ascompared to vehicle treatment, confirming the in vivo efficacy ofWX-039. The tumor growth inhibition rate assessed during the experimentis shown in FIG. 43B. As shown in FIG. 43C, there was no substantialbody weight change caused by WX-039 during the experiment, indicatingthat WX-039 has tolerable toxicity.

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein. To theextent those references contradict or are inconsistent with anystatements in this application, the text of the application willcontrol. The disclosure will employ, unless otherwise indicated,conventional techniques of immunology, molecular biology and cellbiology, and pathology, which are well known in the art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the inventions described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

1. (canceled)
 2. A method of manufacturing a polypeptide having a lengthof 7-14 amino acids, wherein the polypeptide is derived from the HD2domain of human B-cell CLL/lymphoma 9 (BCL9), the polypeptide is capableof undergoing a reaction to form a hydrocarbon crosslinker, and thepolypeptide comprises any sequence selected from: (SEQ ID NO: 79)LQTLRXaa₁IQRXaa₂L; and (SEQ ID NO: 82) Xaa₁LQXaa₂LRXaa₃IQRXaa₄L

and wherein: Xaa₁, Xaa₂, Xaa₃, and Xaa₃ are each an α-methyl, α-alkenylamino acid; the method comprising using solid phase synthesis togenerate the polypeptide.
 3. The method of claim 2, wherein theα-methyl, α-alkenyl amino acid is selected from(S)-2-(4′-pentenyl)alanine, (R)-2-(4′-pentenyl)alanine,(S)-2-(7′-octenyl)alanine, and (R)-2-(7′-octenyl)alanine.
 4. The methodof claim 2, further comprising manufacturing a polypeptide having alength of 7-14 amino acids, wherein the polypeptide is derived from theHD2 domain of human B-cell CLL/lymphoma 9 (BCL9), the polypeptidecomprises a hydrocarbon crosslinker, and the polypeptide comprises anysequence selected from: (SEQ ID NO: 79) LQTLRXaa₁IQRXaa₂L; and(SEQ ID NO: 82) Xaa₁LQXaa₂LRXaa₃IQRXaa₄L

and wherein: a) Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are each alanine; b) a firsthydrocarbon crosslinker is present between Xaa₁ and Xaa₂; and/or c) asecond hydrocarbon crosslinker is present between Xaa₃ and Xaa₄; themethod comprising forming the one or more of said hydrocarboncrosslinkers by metal-mediated ring-closing olefin metathesis of thepolypeptide manufactured in claim
 2. 5. The method of claim 4, furthercomprising purifying the polypeptide by high-performance liquidchromatography (HPLC).
 6. The method of claim 5, wherein the purifiedpolypeptide is substantially free of metal.
 7. The method of claim 4,wherein the hydrocarbon crosslinker is selected from—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂— and—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—.
 8. The method of claim 4,wherein the hydrocarbon crosslinker has an S-configuration on at leastone end, an R-configuration on at least one end, or has anS-configuration on one end and an R-configuration on the other end. 9.The method of claim 4, wherein: a) the polypeptide consists of an aminoacid sequence of: (SEQ ID NO: 79) LQTLRXaa₁IQRXaa₂L;

b) Xaa₁ and Xaa₂ are each alanine; c) a hydrocarbon crosslinker ispresent between Xaa₁ and Xaa₂; d) the hydrocarbon crosslinker is—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—; and e) the hydrocarbon crosslinker hasan S-configuration at both ends.
 10. The method of claim 4, wherein: a)the polypeptide consists of an amino acid sequence of (SEQ ID NO: 82)Xaa₁LQXaa₂LRXaa₃IQRXaa₄L;

b) Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are each alanine; c) a first hydrocarboncrosslinker is present between Xaa₁ and Xaa₂; d) a second hydrocarboncrosslinker is present between Xaa₃ and Xaa₄; e) the first hydrocarboncrosslinker is —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—; f) the secondhydrocarbon crosslinker is —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—; g) the firsthydrocarbon crosslinker has an S-configuration at one end and anR-configuration at the other end; and h) the second hydrocarboncrosslinker has an S-configuration at both ends.
 11. The method of claim4, wherein the N-terminus is optionally modified with an acetyl group;and C-terminus of the polypeptide is modified with NH₂, two units ofβ-alanine, 2-Naphthylalanine, or 2-Naphthylalanine linked to two unitsof β-alanine, wherein the carboxyl group of the C-terminus modificationis further modified with NH₂.